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

Abstract

The present disclosure provides adeno-associated viral (AAV) vectors, pharmaceutical compositions, methods of manufacture, and methods for delivering the same to a subject comprising coevolved capsid variant proteins.

Description

[0001] FEDERALLY SPONSORED RESEARCH

[0002] This application was made with government support under federal grant numbers R01 HL089221 and UG3 AR075336, both awarded by the National Institutes of Health. The federal government has certain rights in the application.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application Serial No. 63 / 020,062, filed May 5, 2020, which is incorporated by reference herein in its entirety for all purposes. TECHNICAL FIELD

[0005] The present disclosure relates to modified capsid proteins from adeno-associated virus (AAV) and viral capsids and viral vectors comprising the same. In particular, the present disclosure relates to modified AAV capsid proteins and capsids comprising the same that can be incorporated into viral vectors to enable expression in any cell or tissue type of a mammalian subject.

[0006] INCORPORATION BY REFERENCE OF ELECTRONICALLY FILED SEQUENCE LISTING

[0007] The electronic version of the sequence listing is filed concurrently with the application and is incorporated herein by reference in its entirety. The electronic file contains 611 kilobytes and is named 21-2066-WO_SequenceListing_ST25.txt. BACKGROUND

[0008] Adeno-associated virus (AAV) vectors have become a leading platform for gene therapy to treat a variety of diseases. Despite clinical successes using AAV-based gene therapies, limitations and challenges associated with using this gene delivery platform remain. For example, the efficacy of gene therapy with vectors (viral or non-viral) is sometimes reduced due to an immune response by the subject to the gene-carrying vector. Additionally, the route of administration must be optimized to ensure delivery to the target tissue(s) in the subject. This is particularly true in treating disorders of the central nervous system (CNS) and the peripheral nervous system (PNS). When administered systemically, the blood-brain barrier can impede AAV-based therapies from entering the CNS, and direct administration to CNS tissue can involve invasive surgery. Furthermore, high doses of AAV-based therapies necessary to produce sufficient transduction of target CNS and PNS tissues increase the risk of side effects and / or undesirable immune responses. Additionally, the need to produce high doses of AAV creates a manufacturing burden.

[0009] The known AAV serotypes each have specific tissue tropism, and there are some tissues (e.g., kidney) that cannot be readily targeted using these AAVs. Additionally, AAV transduction in systemic organs (e.g., heart, liver, or lung) can vary significantly for a given dose in various model organisms used during clinical development (e.g., dogs, pigs, non-human primates) and in human subjects. This inability to accurately test AAV-based therapies in animal models prior to human use is also problematic.

[0010] As the scope of AAV gene transfer applications expands, including advances in gene therapy for CNS and / or PNS disorders, there remains a need in the art to address the differences in AAV tropism across different species. These differences often lead to non-linear vector dose biodistribution relationships - subsequently affecting clinical translation - when scaling animal models from small to large. Thus, there is a need in the art to develop AAV gene delivery platforms with greater translatability across multiple species. Additionally, there is a need to develop AAV-based gene therapies that can selectively and specifically target tissues of interest, including tissues that are difficult to target using known AAV serotypes. SUMMARY

[0012] The present disclosure provides, at least in part, methods and compositions comprising adeno-associated virus (AAV) capsid proteins comprising one or more amino acid substitutions, wherein the substitutions introduce one or more improved functionalities into AAV vectors comprising these modified capsid proteins, such as, but not limited to, the ability to evade host antibodies, selective tropism, and / or higher transduction efficiency.

[0013] One aspect of the present disclosure provides a recombinant AAV vector comprising an AAV capsid protein variant disclosed herein. In some embodiments, the recombinant AAV vector herein can 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 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, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28. In some embodiments, the recombinant AAV vector herein can comprise an AAV capsid protein variant, wherein the capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0014] In some embodiments, the recombinant AAV vectors herein can comprise an AAV capsid protein variant, wherein the capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28; and wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0015] In some embodiments, the recombinant AAV vectors herein can 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 1-10, 11-20, 20-30, or 30-50 amino acid substitutions relative thereto.

[0016] 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: 2-19.

[0017] In some embodiments, the AAV capsid protein variant herein has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28. In some embodiments, the AAV capsid protein variant herein has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0018] In some embodiments, the AAV capsid protein variant herein can have at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 can be substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28; and wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 can be substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0019] In some embodiments, the AAV capsid protein variants herein can have the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence that is at least 90% or at least 95% identical thereto. In some embodiments, the capsid protein variants herein can have the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence that has 1-10, 11-20, 20-30, or 30-50 amino acid substitutions relative thereto.

[0020] Another aspect of the present disclosure provides pharmaceutical compositions comprising any of the AAV capsid protein variants and / or AAV vectors disclosed herein. In some embodiments, the pharmaceutical compositions herein can further comprise at least one pharmaceutically acceptable carrier.

[0021] Another aspect of the present disclosure provides methods of introducing a recombinant AAV vector into a target cell. In some embodiments, the methods of introducing a recombinant AAV vector into a target cell herein can comprise contacting a target cell with any of the recombinant AAV vectors (e.g., ccAAV) and / or pharmaceutical compositions disclosed herein. In some embodiments, the methods herein can deliver one or more therapeutic heterologous molecules to a target cell in a subject, the method comprising administering to the subject any of the recombinant AAV vectors (e.g., ccAAV) and / or pharmaceutical compositions disclosed herein. In some embodiments, any of the recombinant AAV vectors (e.g., ccAAV) and / or pharmaceutical compositions disclosed herein can be administered to a subject by intramuscular injection, intravenous injection, intracoronary injection, intraarterial injection, or any combination thereof.

[0022] Another aspect of the present disclosure provides methods of evolving new adeno-associated virus strains comprising passaging AAV capsid libraries across multiple mammalian species. In some embodiments, the methods herein can utilize AAV capsid libraries comprising AAV capsids packaging different genomes encoding mutagenized capsid gene sequences. In some embodiments, the methods herein can administer the AAV capsid libraries herein to Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primates (Macaca, macaque), or Homo sapiens (human), and any combination or repeat cycle thereof. In some embodiments, the methods herein can enrich for adeno-associated virus (AAV) capsid protein sequences herein by passaging AAV capsid libraries 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 or derived from the group consisting of: spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglion, 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 disclosed herein having improved gene transfer efficiency in any mammalian species selected from the group consisting of: Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primates (Macaca, macaque), or Homo sapiens (human), and any combination or repeat cycle thereof. In some embodiments, the methods herein can produce AAV capsid protein variants as disclosed herein having improved gene transfer efficiency in any cell type or tissue from the group consisting of: spinal cord, dorsal root ganglion, 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 disclosed herein having improved immune response in any cell type or tissue from the group consisting of: spinal cord, dorsal root ganglion, 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 having improved tropism in any cell type or tissue from the group consisting of: spinal cord, dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0023] One aspect of the present disclosure provides kits, wherein the kits can comprise any of the compositions or AAV vectors disclosed herein and at least one container.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The application can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] FIG. 1A and 1B Bubble plots illustrating analysis of library diversity, directed evolution, and enrichment of AAVs comprising capsid proteins with novel peptide substitutions according to certain embodiments herein. High-throughput sequencing was performed on the parental (A) FIG. 1A ) and evolved libraries from three rounds of evolution (B) FIG. 1B ) using the lllumina MiSeq platform. After analysis with custom Perl scripts, enriched amino acid sequences were plotted. Each bubble represents a different capsid protein amino acid sequence, with the radius of the bubble proportional to the number of reads for that variant in the corresponding library. The y-axis represents the percentage of total reads from the sequencing run. To facilitate visualization, the data is expanded along the x-axis. The reduction in the percentage of unique clones (96.5%) directly demonstrates the removal of many "mismatched" sequences after the first and / or second round of evolution. Dominant segregants were selected for further analysis. As shown in FIG. 1B , after three rounds of evolution in three different species, next-generation sequencing revealed that the capsid protein of AAVcc47 was the most enriched amino acid sequence (i.e., clone) in the AAVVR4 library.

[0027] FIGS. 2A-2D Illustration of mCherry reporter gene expression in the heart of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscope images of mCherry expression in heart vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (A) FIG. 2A ) or AAV.cc47 (B) FIG. 2B ) are shown. FIG. 2C Graphs depicting corrected total cellular fluorescence of a series of multiple images are shown. FIG. 2D Graphs depicting vector biodistribution in infected mouse hearts are shown.

[0028] FIGS. 3A-3C Illustration of mCherry reporter gene expression in the skeletal muscle of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscope images of mCherry expression in skeletal muscle vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (A) FIG. 3A ) or AAV.cc47 (B) FIG. 3B ) are shown. FIG. 3C Graphs depicting corrected total cellular fluorescence of a series of multiple images are shown.

[0029] FIGS. 4A-4D Illustration of mCherry reporter gene expression in the liver of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscope images of mCherry expression in liver sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (A) FIG. 4A ) or AAV.cc47 (B) FIG. 4BRepresentative fluorescent microscope images of mCherry expression in liver vibratome sections 24 hours after fixation with 4% PFA. FIG. 4C Graphs showing corrected total cell fluorescence depicting a series of multiple images. FIG. 4D Graphs showing depicting vector biodistribution in the livers of infected mice.

[0030] FIGS. 5A-5C Illustrates mCherry reporter gene expression in the kidneys of C57 / B6 mice according to certain embodiments herein. Representative fluorescent microscope images of mCherry expression in kidney vibratome sections 24 hours after fixation with 4% PFA of mice infected with AAV9 ( FIG. 5A ) or AAV.cc47 ( FIG. 5B ). FIG. 5C Graphs showing corrected total cell fluorescence depicting a series of multiple images.

[0031] FIGS. 6A-6D Illustrates GFP reporter gene expression in the hearts of C57 / B6 mice according to certain embodiments herein. Representative fluorescent microscope images of GFP expression in heart vibratome sections 24 hours after fixation with 4% PFA of mice infected with AAV9 ( FIG. 6A ) or AAV.cc81 ( FIG. 6B ) or AAV.cc84 ( FIG. 6C ). FIG. 6D Graphs showing corrected total cell fluorescence depicting a series of multiple images.

[0032] FIGS. 7A-7C Illustrates GFP reporter gene expression in the skeletal muscle of C57 / B6 mice according to certain embodiments herein. Representative fluorescent microscope images of GFP expression in skeletal muscle vibratome sections 24 hours after fixation with 4% PFA of mice infected with AAV9 ( FIG. 7A ) or AAV.cc81 ( FIG. 7B ). FIG. 7C Graphs showing corrected total cell fluorescence depicting a series of multiple images.

[0033] FIGS. 8A-8D Illustrates GFP reporter gene expression in the livers of C57 / B6 mice according to certain embodiments herein. Representative fluorescent microscope images of GFP expression in liver vibratome sections 24 hours after fixation with 4% PFA of mice infected with AAV9 ( FIG. 8A ), AAV.cc481 ( FIG. 8B ) or AAV.cc84 ( FIG. 8C ). FIG. 8D Graphs showing corrected total cell fluorescence depicting a series of multiple images.

[0034] FIGS. 9A-9C This demonstrates GFP reporter gene expression in the kidneys of C57 / B6 mice according to certain embodiments described herein. Infection with AAV9 (…) FIG. 9A ) or AAV.cc81 ( FIG. 9B Representative fluorescence microscopy images of GFP expression in kidney sections obtained by vibratory slicer from mice 24 hours after fixation with 4% PFA. FIG. 9C A graph depicting corrected total cellular fluorescence across a series of multiple images is shown.

[0035] FIGS. 10A-10E This describes the expression of fluorescent reporter genes in the brain regions of C57 / B6 mice as assessed by immunohistochemistry (IHC) according to certain embodiments described herein. FIG. 10A The brain regions from mice with pseudo-infection were depicted, and FIG. 10B Delineating brain regions from mice infected with the AAV9 vector. FIG. 10C Delineating brain regions from mice infected with AAV.cc47. FIG. 10D Brain regions from mice infected with AAV.cc81 were depicted, and FIG. 10E Brain regions from mice infected with AAV.cc84 are depicted. The brain regions shown include: Ctx = cerebral cortex; Hc = hippocampus; Cb = cerebellum; Th = thalamus; Str = striatum; and mb = mushroom body.

[0036] FIGS. 11A-11G This describes the AVV.cc47 transduction in pig brain regions as assessed by immunohistochemistry (IHC) according to certain implementation schemes described herein. FIG. 11A IHC staining of mCherry in porcine frontal cortex was described. FIG. 11B IHC staining of mCherry in the cortex of the apical lobe of pigs was described. FIG. 11C IHC staining of mCherry in the porcine parietal thalamus was described. FIG. 11D IHC staining of mCherry in the cortex of pig occipital leaf was depicted. FIG. 11E IHC staining of mCherry in porcine brainstem is described. FIG. 11F IHC staining of mCherry in the porcine cerebellum is described. FIG. 11G IHC staining of mCherry in the porcine midbrain is described.

[0037] FIGS. 12A-12G This describes the AVV.cc84 transduction in pig brain regions as assessed by immunohistochemistry (IHC) according to certain implementation schemes described herein. FIG. 12A IHC staining of GFP in porcine frontal cortex is described. FIG. 12B IHC staining of GFP in the cortex of porcine parietal leaves was described. FIG. 12CIHC staining of GFP in the thalamus of a pig is described. FIG. 12D IHC staining of GFP in the occipital cortex of a pig is described. FIG. 12E IHC staining of GFP in the brainstem of a pig is described. FIG. 12F IHC staining of GFP in the cerebellum of a pig is described. FIG. 12G IHC staining of GFP in the midbrain of a pig is described.

[0038] FIGS. 13A-13F Illustration of AAV.cc47 and AVV.cc84 transduction in the spinal cord of a pig according to certain embodiments herein. Sections of the spinal cord of a pig were subjected to IHC staining for AVV.cc47 ( FIG. 13A ) and AVV.cc84 ( FIG. 13B ). mCherry fluorescence was measured in the white matter ( FIG. 13C ) and the gray matter ( FIG. 13E ) to assess AVV.cc47. GFP fluorescence was measured in the white matter ( FIG. 13D ) and the gray matter ( FIG. 13F ) to assess the presence of AVV.cc84.

[0039] FIGS. 14A-14F Illustration of AAV.cc47 and AVV.cc84 transduction in the heart and liver of a pig according to certain embodiments herein. AVV.cc47 transduction was assessed by IHC staining for mCherry in the left ventricle of the pig heart ( FIG. 14A ), the right ventricle of the pig heart ( FIG. 14B ), and the liver ( FIG. 14C ). AVV.cc84 transduction was assessed by IHC staining for GFP in the left ventricle of the pig heart ( FIG. 14D ), the right ventricle of the pig heart ( FIG. 14E ), and the liver ( FIG. 14F ).

[0040] FIGS. 15A-15E Illustration of AAV9 and AVV.cc47 transduction in the heart and liver of a non-human primate according to certain embodiments herein. AVV9 transduction was assessed by IHC staining for mCherry in the liver ( FIG. 15A ) and the heart ( FIG. 15C ) of a non-human primate. AVV.cc47 transduction was assessed by IHC staining for mCherry in the liver ( FIG. 15B ) and the heart ( FIG. 15D ) of a non-human primate. FIG. 15E Showed biodistribution of recombinant AAV in non-human primates.

[0041] FIGS. 16A-16DIllustrates AAV9, AAV.cc47, and AAV.cc84 transduction in the non-human primate brain according to certain embodiments herein. AAV9 transduction was assessed by IHC staining for mCherry in the non-human primate brain FIG. 16B ), AAV.cc47 transduction was assessed by IHC staining for mCherry FIG. 16C ), and AAV.cc84 transduction was assessed by IHC staining for GFP FIG. 16D ). FIG. 16A Brain sections from a sham-injected control non-human primate are shown.

[0042] FIGS. 17A-17E Illustrates validation of AAVcc47 cardiac transduction according to certain embodiments herein. FIG. 17A Human iPSC cardiomyocytes transduced with AAV9 or cc47 packaging GFP driven by the Cbh promoter are shown. FIG. 17B Quantification of GFP+ area percentage in multiple images of ( FIG. 17A ). FIG. 17C IV injection of AAV9 or AAVcc47 packaging CBh:GFP in a human cardiac patch mouse model is shown. FIG. 17D Fluorescent imaging of cardiac patches is shown. FIG. 17E Immunofluorescence of troponin T (red) and GFP (green) is shown following i.v. administration of GFP-delivering AAV9 and AAVcc47 under the control of an injury-inducible promoter following myocardial infarction.

[0043] FIGS. 18A-18E Illustrates representative images of native tdTomato fluorescence in mouse hearts following i.v. administration of mock treatment ( FIG. 18A ), AAV9 ( FIG. 18B ), AVV.cc47 ( FIG. 18C ), and AVV.cc84 ( FIG. 18D ) according to certain embodiments herein. FIG. 18E Biodistribution of recombinant AAV in mouse hearts is shown.

[0044] FIGS. 19A-19E Illustrates representative images of native tdTomato fluorescence in mouse livers following i.v. administration of mock treatment ( FIG. 19A ), AAV9 ( FIG. 19B ), AVV.cc47 ( FIG. 19C ), and AVV.cc84 ( FIG. 19D ) according to certain embodiments herein. FIG. 19E Biodistribution of recombinant AAV in mouse livers is shown.

[0045] FIGS. 20A-20ERepresentative images of native tdTomato fluorescence in the lungs of mice following i.v. administration of mock treatment FIG. 20A , AAV9 ( FIG. 20B ), AVV.cc47 ( FIG. 20C ), and AVV.cc84 ( FIG. 20D ) according to certain embodiments herein. FIG. 20E Biodistribution of recombinant AAV in the liver of mice is shown.

[0046] FIGS. 21A-21D Demonstration of CRISPR / Cas9 gene editing with ccAAV vectors according to certain embodiments herein. FIG. 21A Demonstration of the dual vector strategy used herein, using a first vector and a second vector, where the first vector has a truncated CB promoter driving SaCas9 and a U6 promoter driving one sgRNA, and the second vector has the same design as the second sgRNA. FIG. 21B Native tdTomato fluorescence in the liver and heart of Ai9 mice following administration of AAV9 or cc47 at a dose of 2el2vg / kg is shown. FIG. 21C Gene editing efficiency determined by counting the total number of tdTomato+ cells and dividing by the total number of DAPI+ cells is shown. FIG. 21D PCR editing assay is shown, where the unedited band ( 1160bp) and edited band ( 270bp) are recorded. p <0.01.

[0047] FIGS. 22A-22C Demonstration of validation of CRISPR / Cas9 gene editing with ccAAV vectors according to certain embodiments herein. FIG. 22A Ai9 livers were sectioned and imaged for native tdTomato expression is shown. FIG. 22B Graph depicting quantification of gene editing efficiency by counting the total number of tdTomato+ cells and normalizing to the total number of DAPI+ cells is shown. FIG. 22C Ai9 hearts were sectioned and imaged for native tdTomato expression. Both tissues were cryo-sectioned into 14pm thick sections.

[0048] FIGS. 23A-23F Quantification of CRISPR / Cas9 by measuring fluorescence intensity according to certain embodiments herein is shown. Fluorescence intensity was measured from multiple images to quantify native tdTomato expression in Ai9 mice injected with AAV9 vector or AAV.cc47 vector. FIG. 23A Corrected total cell fluorescence plot in the livers of all injected Ai9 mice is shown.FIG. 23B Graph showing corrected total cellular fluorescence in livers of all injected female Ai9 mice. FIG. 23C Graph showing corrected total cellular fluorescence in hearts of all injected Ai9 mice. FIG. 23D Graph showing corrected total cellular fluorescence in hearts of all injected female Ai9 mice. FIG. 23E Graph showing corrected total cellular fluorescence in livers of all injected male Ai9 mice. FIG. 23F Graph showing corrected total cellular fluorescence in hearts of all injected male Ai9 mice. P values <0.05; NS = not significant.

[0049] FIG. 24A and 24B Illustrates quantification of CRISPR / Cas9 by measuring relative PCR band intensity according to certain embodiments herein. FIG. 24A Graph showing PCR band intensity (relative to mock, unedited band) of PCR bands resulting from PCR editing assay of liver tissue of Ai9 mice injected with AAV9 vector or AAV.cc47 vector. FIG. 24B Graph showing PCR band intensity (relative to mock, unedited band) of PCR bands resulting from PCR editing assay of heart tissue of Ai9 mice injected with AAV9 vector or AAV.cc47 vector.

[0050] FIG. 25A and 25B Illustrates quantification of CRISPR / Cas9 gene editing efficiency in liver and heart according to certain embodiments herein. FIG. 25A Graph showing percent gene editing efficiency of liver tissue from Ai9 mice injected with AAV9 vector or AAV.cc47 vector. FIG. 25B Graph showing percent gene editing efficiency of heart tissue of Ai9 mice injected with AAV9 vector or AAV.cc47 vector.

[0051] FIG. 26A and 26B Illustrates mCherry reporter gene expression in hearts of C57 / B6 mice according to certain embodiments herein. FIG. 26A Graph showing representative fluorescence microscope images showing mCherry expression in heart vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 or AAV.cc44. FIG. 26B Graph showing corrected total cellular fluorescence depicting a series of multiple images.

[0052] FIG. 26C and 26DIllustrates mCherry reporter gene expression in skeletal muscle of C57 / B6 mice according to certain embodiments herein. FIG. 26C Representative fluorescence microscope images showing mCherry expression in skeletal muscle vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 or AAV.cc44 are shown. FIG. 26D Plots depicting corrected total cellular fluorescence for a series of multiple images are shown.

[0053] FIG. 27A and 27B Illustrates mCherry reporter gene expression in liver of C57 / B6 mice according to certain embodiments herein. FIG. 27A Representative fluorescence microscope images showing mCherry expression in liver vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 or AAV.cc44 are shown. FIG. 27B Plots depicting corrected total cellular fluorescence for a series of multiple images are shown.

[0054] FIG. 27C and 27D Illustrates mCherry reporter gene expression in kidney of C57 / B6 mice according to certain embodiments herein. FIG. 27C Representative fluorescence microscope images showing mCherry expression in kidney vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 or AAV.cc44 are shown. FIG. 27D Plots depicting corrected total cellular fluorescence for a series of multiple images are shown.

[0055] FIGS. 28A-28C Illustrates fluorescent reporter gene expression in brain regions of C57 / B6 mice assessed by immunohistochemistry (IHC) according to certain embodiments herein. FIG. 28A Brain regions from mock infected mice are depicted while FIG. 28B Brain regions from mice infected with AAV9 vector are depicted while FIG. 28C Brain regions from mice infected with AAV.cc44 are depicted. Brain regions shown include: Ctx = cerebral cortex; Hc = hippocampus; Cb = cerebellum; Th = thalamus; Str = striatum; and mb = mushroom bodies.

[0056] FIG. 29A and 29B Illustrates a schematic of an AAV vector for administration according to certain embodiments herein. FIG. 29ARecombinant capsid proteins produced as vectors packaging CBh-mCherry (AAV.cc47 and AAV.cc44) with variable region 4 (VR4) on the highlighted capsid surface of the full capsid (top panel) and (bottom panel). FIG. 29B Recombinant capsid proteins produced as vectors packaging CBh-eGFP (AAV.cc81 and AAV.cc84) with variable region 8 (VR8) on the highlighted capsid surface of the full capsid (top panel) and (bottom panel).

[0057] FIGS. 30A-30F Representative images of mCherry or eGFP expression in the mouse brain following intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein are illustrated. FIG. 30A Full mouse brain and selected brain regions following ICV injection of AAV9 vector (mCherry) are depicted. FIG. 30B Full mouse brain and selected brain regions following ICV injection of AAV.cc44 vector (mCherry) are depicted. FIG. 30C Full mouse brain and selected brain regions following ICV injection of AAV.cc47 vector (mCherry) are depicted. FIG. 30D Full mouse brain and selected brain regions following ICV injection of AAV9 vector (eGFP) are depicted. FIG. 30E Full mouse brain and selected brain regions following ICV injection of AAV.cc81 vector (eGFP) are depicted. FIG. 30F Full mouse brain and selected brain regions following ICV injection of AAV.cc84 vector (eGFP) are depicted. Brain regions shown include: Ctx = cerebral cortex; Hc = hippocampus; and Cb = cerebellum.

[0058] FIGS. 31A-31E Representative images and figures of eGFP expression in the mouse brain following intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein are illustrated. FIG. 31A Images of selected brain regions following immunofluorescence (IF) staining of DAPI, eGFP, and NeurN in brain tissue collected and processed following ICV injection of AAV9 vector (eGFP) are depicted. Images from all three stains are merged to show co-localization. FIG. 31B Images of selected brain regions following immunofluorescence (IF) staining of DAPI, eGFP, and NeurN in brain tissue collected and processed following ICV injection of AAV.cc84 vector (eGFP) are depicted. Images from all three stains are merged to show co-localization. In the cerebellum (CB, FIG. 31C ), hippocampus (HC, FIG. 31D ), and cerebral cortex (CTX, FIG. 31EQuantification of the number of neurons with eGFP and NeurN staining in the

[0059] FIGS. 32A-32E Representative images and graphs of mCherry expression in the mouse brain following intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein are illustrated. FIG. 32A Images of selected brain regions following immunofluorescence (IF) staining of DAPI, mCherry, and NeurN in brain tissue harvested and processed following ICV injection of AAV9 vector (mCherry) are described. Images from all three stains were merged to show co-localization. FIG. 32B Images of selected brain regions following immunofluorescence (IF) staining of DAPI, eGFP, and NeurN in brain tissue harvested and processed following ICV injection of AAV.cc47 vector (mCherry) are depicted. Images from all three stains were merged to show co-localization. In the cerebellum (CB, FIG. 32C ), hippocampus (HC, FIG. 32D ), and cerebral cortex (CTX, FIG. 32E ), the number of neurons with mCherry and NeurN staining was quantified. DETAILED DESCRIPTION

[0061] Adeno-associated virus (AAV) vectors have become a leading platform for therapeutic gene delivery. Unfortunately, AAV-based gene therapy is sometimes less effective than desired due to difficulties, for example, in optimizing the route of administration to target the cells or tissues of interest and the subject's immune response to the vector carrying the therapeutic gene (e.g., the transgene of interest). Pre-existing host-derived antibodies generated upon natural exposure to AAV or recombinant AAV vectors prevent the first as well as repeated administration of AAV vectors as vaccines and / or for gene therapy. Serological studies have revealed a high prevalence of antibodies in the world population, with about 67% of people having antibodies against AAV1, 72% against AAV2, and about 40% against AAV5-AAV9. In gene therapy, pre-existing antibodies in the subject cause problems because certain clinical situations involving gene silencing or tissue degeneration require multiple administrations of AAV vectors to maintain long-term expression of the transgene.

[0062] The known AAV serotypes each have specific tissue tropism, and there are some tissues (e.g., kidney) that cannot be easily targeted using these AAVs. It is particularly difficult to use AAV vectors to deliver therapeutic genes to treat disorders of the central nervous system (CNS) and peripheral nervous system (PNS) because the blood-brain barrier can prevent AAV-based therapies from reaching the desired target. AAV transduction in systemic organs (e.g., heart, liver, or lung) can vary significantly for a given dose in various model organisms used during clinical development (e.g., dogs, pigs, non-human primates) and in human subjects.

[0063] To avoid these problems, there is a need for recombinant AAV vectors that evade antibody recognition and / or selectively target CNS tissues. The aspects provided in the present disclosure will facilitate a) expanding the eligible population of patients suitable for AAV-based gene therapy and b) allowing multiple, repeated administration of AAV-based gene therapy vectors. In addition, there is a need to develop AAV-based gene therapies that are capable of selectively and specifically targeting tissues of interest, including tissues that are difficult to target using known AAV serotypes, such as the kidney.

[0064] The present disclosure is based, at least in part, on the new discovery that the capsid antigenicity and functional properties (e.g., tropism and transduction) of AAV capsids and capsid proteins overlap in structural context and can be modified to confer improved functionality. Based on the present disclosure, the AAV capsid proteins and adeno-associated virus (AAV) vectors comprising AAV capsid proteins disclosed herein can be co-evolved to induce cross-species compatibility, which is a potentially useful property, enabling the use of a given AAV to be reliably translated from a disease non-human model (e.g., rodent, non-human primate) to human. Accordingly, the present disclosure provides cross-species compatible AAV capsid proteins and AAV vectors comprising the AAV capsid proteins herein, methods of making and methods of using the same. As used herein, “cross-species compatible AAV” can refer to an AAV vector comprising an AAV capsid protein variant having an amino acid sequence with mutations and / or substitutions co-evolved for cross-species compatibility.

[0065] I. DEFINITIONS

[0066] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the preferred embodiments, and specific language will be used to describe the same. It will nevertheless be understood that this is not intended to limit the scope of the present disclosure, which is as set forth in the appended claims, as such alterations and further modifications of the present disclosure are readily apparent to those skilled in the art.

[0067] The articles "a" and "an" as used in this specification mean one or more, i.e., at least one, of the elements it introduces. For example, "an element" means one or more elements and can include more than one element.

[0068] "About" is used to provide flexibility to a numerical range endpoint, given that the value can be slightly above or below the endpoint without affecting the desired result. The term "about" in relation to a numerical value can vary by 5% or less of the numerical value.

[0069] Throughout this specification, unless the context requires otherwise, the word "comprise," or variations such as "comprises" or "comprising," will be understood 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.

[0070] "AND / OR", as used herein, means and encompasses any and all possible combinations of one or more of the associated listed items and the absence of an item in certain combinations.

[0071] Further, the present disclosure contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B, and C, it is specifically intended that any of A, B, or C, or a combination thereof, can be omitted or excluded from the complex and the application specifically claims each of these alternatives.

[0072] Unless otherwise stated in this document, the recitation of numerical ranges by endpoints herein is intended to include all numbers and values subsumed within the range. For example, a range of 1 to 50% is intended to include all numbers such as 2 to 40, 10 to 30, or 1 to 3, and the like. These are only examples of what is specifically intended to be encompassed by a given range. Nevertheless, it is specifically intended that the end value of a range include the value of the end point itself. For example, a range of 1 to 5 is specifically intended to include 5. Likewise, a range of 1 to 10% is specifically intended to include 10%.

[0073] 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 type 3A and type 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 rh32.33, AAV rh8, AAV 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, vol. 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A variety of AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-: 375-383; and Table 1).

[0074] The genomic sequences of various different serotypes of AAV and self-replicating parvoviruses, as well as the sequences of the natural terminal repeat sequences (TRs), Rep proteins, and capsid subunits, are known in the art. These sequences can be found in the literature or in public databases (e.g., GenBank). See, e.g., GenBank Accession Nos. 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, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; the disclosures of which are incorporated herein by reference to teach parvovirus and AAV nucleic acid and amino acid sequences. See also Table 1.

[0075] Table 1.

[0076]

[0077]

[0078] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid" are used interchangeably herein and refer to a sequence that is not naturally found in the virus. Typically, the heterologous nucleic acid comprises an open reading frame that encodes a polypeptide of interest or an untranslated RNA (e.g., for delivery to a cell or subject).

[0079] As used herein, "polynucleotide" refers to a sequence of nucleotide bases and can be RNA, DNA, or a DNA-RNA hybrid sequence (including naturally-occurring and non- naturally-occurring nucleotides), but in representative embodiments is a single- or double- stranded DNA sequence.

[0080] As used herein, the term "peptide" refers to a short sequence of amino acids. The term peptide can be used to refer to a portion or region of an AAV capsid amino acid sequence. The 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. An AAV peptide that naturally occurs in an AAV capsid can be replaced with a non-naturally occurring peptide. For example, a non-naturally occurring peptide can be substituted into an AAV capsid to provide a modified capsid such that a naturally occurring peptide is replaced with a non-naturally occurring peptide. Unless otherwise specified, the term "polypeptide" as used herein encompasses peptides and proteins.

[0081] As used herein, the term "amino acid" encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids. Alternatively, the amino acids herein can be modified amino acid residues and / or can be amino acids modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). Naturally occurring L- amino acids are shown in Table 2.

[0082] Table 2

[0083]

[0084] Alternatively, the amino acids can be modified amino acid residues (non-limiting examples are shown in Table 3) and / or can be amino acids modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

[0085] Table 3

[0086]

[0087]

[0088] In addition, non-naturally occurring amino acids can be "unnatural" amino acids (as described in 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.

[0089] As used herein, the term "viral vector," "vector," or "gene delivery vector" refers to a viral (e.g., AAV) particle used as a vehicle for nucleic acid delivery, and it includes a vector genome (e.g., viral DNA [vDNA]) packaged within the viral particle. Alternatively, in some contexts, the term "viral vector" can be used to refer to the vector genome / vDNA alone.

[0090] As used herein, an "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that comprises one or more heterologous nucleic acid sequences. An rAAV vector typically requires only the terminal repeat sequences (TR(s)) in cis to produce a virus. All other viral sequences are non-essential and can be provided in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, an rAAV vector genome will retain only one or more TR sequences in order to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and non-structural protein coding sequences can be provided in trans (e.g., from a vector such as a plasmid or by stably integrating the sequences into a packaging cell). In embodiments of the application, an rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), optionally two TRs (e.g., two AAV TRs), which are typically located at the 5' and 3' ends of the vector genome and flank the heterologous nucleic acid, but need not be contiguous therewith. The TRs can be the same or different from each other.

[0091] The term "terminal repeat sequence" or "TR" includes any viral terminal repeat sequence or synthetic sequence that forms a hairpin structure and can function as an inverted terminal repeat (i.e., mediate a desired function, such as replication, viral packaging, integration, and / or provirus rescue, etc.). 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., the SV40 hairpin used as the SV40 origin of replication) can be used as a TR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. In addition, the TR can be partially or completely synthetic, such as the "double-D sequence" described in U.S. Patent 5,478,745 to Samulski et al.

[0092] An "AAV terminal repeat sequence" 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 sequence need not have a native terminal repeat sequence (e.g., a native AAV TR sequence can be altered by insertion, deletion, truncation, and / or missense mutation), so long as the terminal repeat sequence mediates a desired function, e.g., replication, viral packaging, integration, and / or provirus rescue, etc.

[0093] An AAV vector typically comprises a protein-based capsid and a nucleic acid encapsidated by the capsid. The nucleic acid can be, for example, a vector genome comprising a transgene flanked by inverted terminal repeat sequences. An AAV "capsid" is a roughly spherical protein shell comprising individual "capsid proteins" or "subunits." An AAV capsid typically comprises about 60 capsid protein subunits, 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, wherein the capsid comprises one or more AAV capsid proteins (i.e., subunits). Also described herein are "virus-like particles" or "VLPs," which refer to a capsid that does not comprise any vector genome or nucleic acid comprising a transgene.

[0094] The viral vectors of the present disclosure can also 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, the disclosure of which is incorporated herein by reference in its entirety.

[0095] The viral vectors of the present disclosure can also be double-stranded parvovirus particles as described in International Patent Publication WO 01 / 92551, the disclosure of which is incorporated herein by reference in its entirety. Thus, in some embodiments, a double-stranded (duplexed) genome can be packaged into the viral capsid of the present invention. Furthermore, the viral capsid or genome elements can contain other modifications, including insertions, deletions, and / or substitutions.

[0096] The term "self-complementary AAV" or "scAAV" refers to a recombinant AAV vector that forms a self-annealing, duplex inverted repeat DNA molecule, resulting in earlier and more robust transgene expression compared to conventional single-stranded (ss) AAV genomes. See, e.g., McCarty, D.M., et al., Gene Therapy 8, 1248-1254 (2001). Unlike conventional ssAAV, scAAV can bypass the second strand synthesis, the rate-limiting step for gene expression. In addition, the double-stranded scAAV is less susceptible to DNA degradation after viral transduction, thereby increasing the copy number of stable episomes. Notably, scAAV typically can only accommodate about 2.4 kb of genome, which is half the size of conventional AAV vectors. In some embodiments, the AAV vectors described herein are self-complementary AAV.

[0097] A "therapeutic polypeptide" or "therapeutic protein" is a polypeptide or protein that can alleviate, reduce, prevent, retard, and / or stabilize symptoms resulting from a deficiency or defect in a protein in a cell or subject, and / or is a polypeptide that otherwise imparts a benefit to a subject, such as an anticancer effect or improved graft survival.

[0098] The terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean a reduction in severity, amelioration of, stabilization of, and / or a decrease in at least one clinical symptom and / or a delay in the progression of the disease or disorder in a subject.

[0099] The terms "prevent," "preventing," and "prevention of" (and grammatical variations thereof) mean preventing and / or delaying the onset of a disease, disorder, and / or clinical symptoms in a subject, and / or lessening the severity of the onset of a disease, disorder, and / or clinical symptoms relative to what occurs in the absence of the methods of the present disclosure. Prevention can be complete, i.e., the total absence of a disease, disorder, and / or clinical symptoms. Prevention can also be partial, such that the occurrence and / or severity of a disease, disorder, and / or clinical symptoms in a subject is less than what occurs in the absence of the present disclosure.

[0100] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to humans and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, e.g., mammals and non-mammals, such as 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 therapies.

[0101] A "therapeutically effective" amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively, a "therapeutically effective" amount is an amount that will provide some alleviation, reduction, decrease, or stabilization in at least one clinical symptom of the subject. Those skilled in the art will appreciate that the therapeutic effect need not be complete or partial for the improvement or benefit to be provided to the subject, so long as some benefit is provided to the subject.

[0102] A "prophylactically effective" amount as used herein is an amount that is sufficient to prevent and / or delay onset of a disease, disorder, and / or clinical symptoms in a subject, and / or reduce and / or delay severity of a disease, disorder, and / or clinical symptoms in a subject relative to what would occur in the absence of the methods of the present disclosure. Those skilled in the art will appreciate that the prophylactic level need not be complete for the improvement or benefit to be provided to the subject, so long as some benefit is provided to the subject.

[0103] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0104] II. Cross-species compatible AAV

[0105] Adeno-associated viruses (AAV) are members of the Parvovirus family, which are small, nonenveloped viruses. Wild-type AAV consists of an icosahedral protein capsid surrounding a single-stranded DNA genome. In wild-type AAV, inverted terminal repeat sequences (ITRs) flank the coding nucleotide sequences (e.g., polynucleotides) of non-structural proteins (encoded by Rep genes) and structural proteins (encoded by capsid genes or Cap genes). Rep genes encode non-structural proteins that modulate functions including replication of the AAV genome. CAP genes encode structural proteins VP1, VP2, and / or VP3 that assemble to form the capsid.

[0106] The present disclosure provides recombinant AAV capsid proteins (VP1, VP2, and / or VP3) comprising modifications (e.g., substitutions) in the amino acid sequence relative to wild-type capsid proteins, as well as AAV capsids and AAV vectors comprising the modified AAV capsid proteins. The present inventors have discovered that the modifications disclosed herein can confer one or more desirable properties to viral vectors comprising the AAV capsid protein variants modified 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. Accordingly, the present disclosure addresses some of the limitations associated with conventional AAV vectors.

[0107] In certain embodiments, the AAV vectors herein can be engineered to include one or more capsid protein variants. In some embodiments, the AAV vectors herein can be cross-species compatible vectors, or“ccAAVs.” In some embodiments, the AAV vectors (e.g., ccAAVs) can be engineered to include at least one or more amino acid substitutions, wherein the one or more substitutions can modify one or more antigenic sites on the AAV capsid protein. Modification of the one or more antigenic sites can result in inhibition of antibody binding to the one or more antigenic sites and / or inhibition of neutralization of infectivity of a viral particle comprising the capsid protein variants described herein.

[0108] Accordingly, in some embodiments herein, the 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, modification of the one or more antigenic sites can result in inhibition of antibody binding to the one or more antigenic sites and / or inhibition of neutralization of infectivity of a viral particle comprising the AAV capsid protein. In some embodiments, the modified antigenic sites can prevent antibody binding or recognition or neutralization of the AAV capsid. In some embodiments, the antibody can be IgG (including IgGl, IgG2a, IgG2b, IgG3), IgM, IgE, or IgA. In some embodiments, the modified antigenic sites can prevent antibody binding, recognition, or neutralization of the AAV capsid from different animal species, wherein the animals are humans, dogs, pigs, cows, non-human primates, rodents, cats, or horses.

[0109] In some embodiments, modification of one or more antigenic sites can result in the AAV vectors herein (e.g., ccAAV) 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 certain cells or tissues, optionally followed by expression (e.g., transcription and optionally translation) of sequences carried by the viral genome, e.g., for a recombinant virus, expression of a heterologous nucleic acid of interest, in the cell. In some embodiments, modification of one or more antigenic sites can result in the AAV vectors herein can exhibit tropism for one or more cell types and / or tissues throughout the body of a subject. In some aspects, modification of one or more antigenic sites can result in the AAV vectors herein can exhibit tropism for brain tissue, lung tissue, skeletal muscle tissue, heart tissue, liver tissue, kidney tissue, and / or pancreatic tissue. In some aspects, modification of one or more antigenic sites can result in the AAV vectors herein exhibit tropism 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 aspects, modification of one or more antigenic sites can result in the AAV vectors herein exhibit tropism for the kidney.

[0110] In some embodiments, one or more amino acid modifications (e.g., substitutions and / or deletions) in the capsid protein variants herein can be in one or more antigenic footprints identified by peptide epitope mapping and / or cryo-EM studies of AAV antibody complexes containing AAV capsid proteins. In some embodiments, one or more antigenic sites herein that can undergo one or more amino acid modifications can be a common antigenic motif (CAM) as described in WO2017 / 058892, which is incorporated by reference herein in its entirety.

[0111] In some embodiments, one or more antigenic sites that can undergo one or more amino acid modifications herein can be in the variable region (VR) of an AAV capsid protein. AAV capsids contain 60 copies (in total) of three VPs (VP1, VP2, VP3) encoded by the cap gene and have overlapping sequences. Each VP can contain an eight-stranded b-barrel motif (bB - bI) and / or a-helices (aA) that are conserved in autonomous parvovirus capsids. The structurally variable regions (VRs) can occur in surface loops that connect b-strands, which cluster to produce local variations in the capsid surface. In some embodiments, one or more amino acid modifications herein that modify one or more antigenic sites in an AAV capsid protein variant herein can be in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, VR-IX, or any combination thereof. In some embodiments, one or more antigenic sites can be in the HI loop of an AAV capsid protein variant herein.

[0112] In some embodiments, an AAV vector (e.g., ccAAV) herein can comprise (i) an AAV capsid protein variant disclosed herein, and (ii) a cargo nucleic acid encapsidated by the capsid protein. According to these embodiments, an AAV vector (e.g., ccAAV) comprising an AAV capsid protein variant described herein can have the following phenotypes: evasion of neutralizing antibodies; enhanced or maintained transduction efficiency; selective tropism for one or more cell and / or tissue types; and any combination thereof.

[0113] In some embodiments, an AAV vector disclosed herein exhibits 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) higher transduction in heart, skeletal muscle, kidney, and brain neurons compared to parental AAV9. In some embodiments, an AAV vector disclosed herein exhibits higher transduction efficiency in some tissue types (e.g., heart, skeletal muscle, kidney, brain neurons) than parental AAV9 and exhibits similar or decreased transduction efficiency in some tissue types (e.g., glial cells) than parental AAV9.

[0114] The present disclosure provides AAVcc.47, which exhibits about 15-fold to about 18-fold higher transduction in heart, skeletal muscle, kidney than parental AAV9. Compared to AAV9, AAVcc.47 has higher transduction in neurons than in brain, while glial cell transduction remains relatively unchanged. AAVcc.81 and AAVcc.84 increase transduction in heart and skeletal muscle by 4-fold, while no significant increase in liver transduction is observed for either ccAAV compared to AAV9. ccAAVs significantly reduce glial cell transduction compared to AAV9, while neuronal tropism is slightly increased. Increasing transduction efficiency by ccAAVs can provide lower dosing regimens of therapeutic vectors.

[0115] In some embodiments, the AAV capsid protein variants disclosed herein can comprise at least one or more amino acid substitutions, wherein from about 1 amino acid residue to 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) can be substituted from the amino acid residues comprising the naturally occurring capsid protein amino acid sequence. According to some embodiments herein, the AAV capsid protein variants herein can have about 7 amino acid residues substituted from the amino acid residues comprising the naturally occurring capsid protein amino acid sequence.

[0116] In some embodiments, the AAV capsid protein variants disclosed herein can have an amino acid sequence that is about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similar to a naturally occurring capsid protein. As used herein, “naturally occurring” or “wild type” refers to that which exists in nature without human modification. In some embodiments, the naturally occurring capsid proteins herein can be derived from a single species. Non-limiting examples of species from which the naturally occurring capsid proteins herein can be derived include those from general 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, fish, and the like. In some embodiments, the species from which the naturally occurring capsid proteins herein can be derived can be Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primates (Macaca, Macaca), or Homo sapiens (human), and any combination thereof. In some embodiments, the AAV capsid protein variants having at least one amino acid substitution as disclosed herein can have an amino acid sequence that is about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similar to a naturally occurring capsid protein having an amino acid sequence referenced by GenBank Accession Nos.: 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, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579, and any combination thereof.

[0117] 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 (Smith & Waterman, Adv. Appl. Math. 2, 482 (1981)), the sequence identity alignment algorithm (Needleman & Wunsch, J Mol. Biol. 48, 443 (1970)), the similarity search method (Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988)), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the best-fit sequence program (as described by Devereux et al., Nucl. Acid Res. 12), or by inspection. Another suitable algorithm is the BLAST algorithm, as described by 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, which is available from Altschul et al., Methods in Enzymology, 266, 460-480 (1996). WU-BLAST-2 uses multiple search parameters, which are optionally set to default values. The parameters are dynamic values and are established by the program itself depending on the composition of the particular sequence and the composition of the particular database against which the sequence of interest is being searched; however, the values can be adjusted to increase sensitivity. In addition, another useful algorithm is gapped BLAST, reported by Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402. For purposes of the present disclosure, percent identity is calculated using the Basic Local Alignment Search Tool (BLAST) available online at blast.ncbi.nlm.nih.gov / Blast.cgi, unless otherwise indicated. Those of skill in the art will appreciate that other algorithms can be substituted as appropriate.

[0118] In some embodiments, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from any of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhIO, AAVIO, 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 can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from a serotype that has known tropism for one or more desired cell and / or tissue types. In some embodiments, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from a serotype that has known tropism for one or more desired human cell and / or tissue types.

[0119] According to these embodiments, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from a serotype that is tropic for the CNS and / or PNS. AAVs can successfully target a variety of different tissue types and cell types within the CNS and PNS, including but not limited to neurons, astrocytes, oligodendrocytes, microglia, Mueller glia, Schwann cells, and satellite cells. In some examples, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein of any AAV serotype that is tropic for astrocytes (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9). In some other embodiments, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein of any AAV serotype that is tropic for oligodendrocytes (e.g., AAV8, AAV9). In some examples, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein of any AAV serotype that is tropic for microglia (e.g., AAV2, AAV5, AAV6, AAV8, AAV9). In some other embodiments, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein of any AAV serotype that is tropic for Mueller glia (e.g., AAV1, AAV2, AAV4, AAV6, AAV8, AAV9). In some examples, the AAV capsid protein variants disclosed herein can have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein of any AAV serotype that is tropic for Schwann cells / satellite glia (e.g., AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9).

[0120] In some embodiments, an AAV capsid protein variant or fragment thereof herein can have an amino acid sequence that is about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similar to a naturally occurring VP1 capsid protein or fragment thereof. In some embodiments, a capsid protein variant herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of any combination of amino acid residues 262-268 of AAV1 (VP1 numbering), or at 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, a capsid protein variant herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of any combination of amino acid residues 370-379 of AAV1 (VP1 numbering), or at 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, a capsid protein variant herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of any combination of amino acid residues 451-459 of AAV1 (VP1 numbering), or at 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, a capsid protein variant herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 472-473 of AAV1 (VP1 numbering), or at 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 can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 493-500 of AAV1 (VP1 numbering), in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 528-534 of AAV1 (VP1 numbering), in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 547-552 of AAV1 (VP1 numbering), in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 588-597 of AAV1 (VP1 numbering), in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2), of amino acid residues 709-710 of AAV1 (VP1 numbering), or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV.In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 262-268 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 370-379 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 451-459 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 472-473 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 493-500 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 528-534 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 547-552 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 588-597 of AAV1 (VP1 numbering); at one or more (e.g., 2) of amino acid residues 709-710 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 716-722 of AAV1 (VP1 numbering); or any combination thereof, or at equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh 10, AAVrh32.33, bovine AAV, or avian AAV.

[0121] In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 262-268 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 370-379 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 451-459 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 472-473 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 493-500 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 528-534 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 547-552 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 588-597 of AAV1 (VP1 numbering); at one or more (e.g., 2) of amino acid residues 709-710 of AAV1 (VP1 numbering); at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 716-722 of AAV1 (VP1 numbering); or any combination thereof, or at equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh 10, AAVrh32.33, bovine AAV, or avian AAV.

[0122] In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region IV (VR4) on the surface of the capsid 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, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region VIII (VR8) on the surface of the capsid 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, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region IV (VR4) on the surface of the capsid 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, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV, and at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region VIII (VR8) on the surface of the capsid 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, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV.

[0123] In some embodiments, the capsid protein variants herein can have at least 90% (e.g., about 90%, 95%, 99%, 100%) sequence identity to the native sequence of AAV9 capsid (SEQ ID NO: 1). In some embodiments, the capsid protein variants herein can comprise amino acid substitutions at any combination of one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region IV (VR4) on the surface of AAV9 capsid. In some embodiments, the capsid protein variants herein can comprise amino acid substitutions at any combination of 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 surface of AAV1 capsid, or at 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 can comprise amino acid substitutions at any combination of 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 surface of AAV9 capsid.

[0124] In some embodiments, the capsid protein variants herein can comprise amino acid substitutions at any combination of one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region VIII (VR8) on the surface of AAV9 capsid. In some embodiments, the capsid protein variants herein can comprise amino acid substitutions at any combination of 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 surface of AAV1 capsid, or at 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 can comprise amino acid substitutions at any combination of 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 surface of AAV9 capsid.

[0125] In some embodiments, the capsid protein variants herein can comprise amino acid substitutions within variable loop region IV (VR4) at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues on the AAV9 capsid surface and within variable loop region VIII (VR8) at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues in any combination. In some embodiments, the capsid protein variants herein can comprise amino acid substitutions within VR4 (452-NGSGQNQ-458 (VP1 numbering; SEQ ID NO: 38)) at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues on the AAV1 capsid surface, or at equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV, in any combination, and within VR8 (586-SAQAQAQ-592 (VP1 numbering; SEQ ID NO: 39)) at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues on the AAV1 capsid surface, or at equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhIO, AAVrh32.33, bovine AAV, or avian AAV, in any combination. In some embodiments, the capsid protein variants herein can comprise amino acid substitutions within VR4 (452-NGSGQNQ-458 (VP1 numbering; SEQ ID NO: 38)) at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues on the AAV9 capsid surface, in any combination, and within VR8 (586-SAQAQAQ-592 (VP1 numbering; SEQ ID NO: 39)) at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues on the AAV9 capsid surface, in any combination.

[0126] In some embodiments, the capsid protein variants herein can have an amino acid sequence that is about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similar to a naturally occurring VP2 capsid protein or fragment thereof from any of the serotypes described herein. In some embodiments, the capsid protein variants herein can comprise amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues in any combination from a naturally occurring VP2 capsid protein or fragment thereof from any of the serotypes described herein.

[0127] In some embodiments, the capsid protein variants herein can have an amino acid sequence that is about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similar to a naturally occurring VP3 capsid protein or fragment thereof from any of the serotypes described herein. In some embodiments, the capsid protein variants herein can comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues in a naturally occurring VP3 capsid protein or fragment thereof from any combination of the serotypes described herein.

[0128] In some embodiments, the AAV vectors (e.g., ccAAV) herein can comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein. According to these embodiments, the AAV vectors (e.g., ccAAV) herein can comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein, wherein the capsid protein comprises a peptide having the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40) at amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1). In some aspects, the AAV vectors herein can comprise an AAV9 capsid protein variant comprising a peptide having the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40) at amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1). In some aspects, the AAV vectors herein can comprise an AAV9 capsid protein variant comprising a peptide having the sequence X 1 may be any amino acid except N; X 2 may be any amino acid except G; X 3 may be any amino acid except S; X 4 may be any amino acid except G; X 5 may be any amino acid except Q; X 6 may be any amino acid except N; and / or X 7 may be any amino acid except Q.

[0129] In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to EGGTVHA (SEQ ID NO: 20). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to FYGTDSA (SEQ ID NO: 21). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to HGQSASR (SEQ ID NO: 22). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to DTPTNQA (SEQ ID NO: 23). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to ITRQAYQ (SEQ ID NO: 24). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to RMFKSNQ (SEQ ID NO: 25). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to GVSLGGG (SEQ ID NO: 26). In some embodiments, a capsid protein variant herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of a native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to KHFLQGE (SEQ ID NO: 27).In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 of native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to MGRERAG (SEQ ID NO: 28).

[0130] In some embodiments, the capsid protein variants herein can share at least about 85% (e.g., about 85%, 90%, 95%, 99%, or 100%) amino acid sequence similarity with any of the sequences set forth in SEQ ID NOs: 2-10. According to some embodiments herein, the capsid protein variants herein comprise any of the sequences set forth in SEQ ID NOs: 2-10. The amino acid sequences of native AAV9 capsid protein (SEQ ID NO: 1) and SEQ ID NOs: 2-10 are provided in Table 4 below.

[0131] In some embodiments, the AAV vectors (e.g., ccAAV) herein can comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein, wherein the capsid protein comprises a peptide having the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125) at amino acids 586-592 of native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering), wherein the peptide is not present in the native AAV9 capsid protein sequence. In some aspects, the AAV vectors herein can comprise an AAV9 capsid protein variant comprising a peptide having the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125), wherein X 1 may be any amino acid except S; X 2 may be any amino acid except A; X 3 may be any amino acid except Q; X 4 may be any amino acid except A; X 5 may be any amino acid except Q; X 6may be any amino acid other than A; and / or X 7 may be any amino acid other than Q.

[0132] In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to LNSSVPS (SEQ ID NO: 29). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to YMDHQVS (SEQ ID NO: 30). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to TSDSLVS (SEQ ID NO: 31). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to NAVGALS (SEQ ID NO: 32). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to MPISHHE (SEQ ID NO: 33). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to DSGARGA (SEQ ID NO: 34). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to NVALALG (SEQ ID NO: 35). In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of the native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to GALRMGM (SEQ ID NO: 36).In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 586-592 of native AAV9 capsid protein (SEQ ID NO: 1) (VP1 numbering) can be substituted with amino acids corresponding to LSGEGAV (SEQ ID NO: 37).

[0133] In some embodiments, the capsid protein variants herein can 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 native AAV9 capsid protein (SEQ ID NO: 1) and SEQ ID NOs: 11-19 are provided in Table 4 below.

[0134] In some embodiments, the AAV vectors (e.g., ccAAV) herein can comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein, wherein the capsid protein comprises a peptide having the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40) at amino acids 452-458 (VP1 numbering) and amino acids 586-592 (VP1 numbering) of native AAV9 capsid protein (SEQ ID NO: 1), wherein the peptide is not present in the native AAV9 capsid protein sequence.

[0135] In some embodiments, the AAV vectors (e.g., ccAAV) herein can comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein, wherein the capsid protein comprises a peptide having the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125) at amino acids 452-458 (VP1 numbering) and amino acids 586-592 (VP1 numbering) of native AAV9 capsid protein (SEQ ID NO: 1), wherein the peptide is not present in the native AAV9 capsid protein sequence.

[0136] In some aspects, the AAV vectors herein can comprise an AAV9 capsid protein variant comprising a peptide having: (1) the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40), wherein X 1 may be any amino acid other than N; X 2 may be any amino acid other than G; X 3 may be any amino acid other than S; X 4 may be any amino acid other than G; X 5 may be any amino acid other than Q; X 6 may be any amino acid other than N; and / or X 7 may be any amino acid other than Q; and (2) the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125), wherein, X 1 may be any amino acid other than S; X 2 may be any amino acid other than A; X 3 may be any amino acid other than Q; X 4 may be any amino acid other than A; X 5 may be any amino acid other than Q; X 6 may be any amino acid other than A; and / or X 7 may be any amino acid other than Q.

[0137] In some embodiments, the capsid protein variants herein can comprise a peptide in which the amino acids corresponding to amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) can be substituted with amino acids corresponding to any one of SEQ ID NOs: 20-28, and the amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) can be substituted with amino acids corresponding to any one of SEQ ID NOs: 29-37.

[0138] In some embodiments, the capsid protein variants herein can share at least about 85% (e.g., about 85%, 90%, 95%, 99%, or 100%) amino acid sequence similarity with any of the sequences set forth in SEQ ID NOs: 46-123. In accordance with some embodiments herein, the capsid protein variants herein comprise any 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.

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] In embodiments where any of the amino acid residues identified as X 1 to X 7 may be the wild-type amino acid residue of the reference amino acid sequence (e.g., AAV9 (SEQ ID NO: 1)). In some embodiments, the capsid protein variants herein can have amino acid substitutions at any combination of residues 452N, 453G, 454S, 455G, 456Q, 457N, and / or 458Q of SEQ ID NO: 1 (AAV9 capsid protein; VP1 numbering). In some embodiments, the capsid protein variants herein can have amino acid substitutions at any combination of residues 586S, 587A, 588Q, 589A, 590Q, 591A, and / or 592Q of SEQ ID NO: 1 (AAV9 capsid protein; VP1 numbering).

[0164] In some embodiments, the capsid protein variants of the present disclosure can be produced by modifying the capsid protein of any AAV capsid protein now known or hereafter discovered. Further, the AAV capsid protein to be modified in accordance with the present disclosure can be a naturally occurring AAV capsid protein (e.g., an AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10, or AAV11 capsid protein or any AAV set forth in Table 1), but is not limited thereto. Those skilled in the art will appreciate that a variety of manipulations of AAV capsid proteins are known in the art and the present disclosure is not limited to modifying naturally occurring AAV capsid proteins. For example, the capsid protein to be modified can 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 hereafter discovered). Such AAV capsid proteins are also within the scope of the present disclosure.

[0165] Some aspects of the 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, which can also be an autonomously replicating parvovirus capsid or a dependovirus capsid. Optionally, the viral capsid herein can be an AAV capsid. In some embodiments, the AAV capsid of the disclosure can be AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV capsids, avian AAV capsids, and / or any other AAV now known or later identified.

[0166] In some embodiments, the modified viral capsids herein can be used as capsid vectors. In some embodiments, a molecule can be packaged by the modified viral capsid and transferred into a cell, where the molecule can include a heterologous DNA, RNA, polypeptide, small organic molecule, metal, or combinations thereof. Heterologous molecules are defined herein as those that are not naturally found in AAV infection, e.g., those not encoded by a wild-type AAV genome. Further, a therapeutically useful molecule for use herein can be associated with the outside of the chimeric viral capsid for transfer of the molecule 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, the therapeutically useful molecules herein can be covalently linked (conjugated or chemically coupled) to the capsid proteins. Methods of covalently linking molecules are known to those of skill in the art.

[0167] In some embodiments, the modified viral capsids herein can be used to generate antibodies against the capsid protein variants disclosed herein. As a further alternative, exogenous amino acid sequences can be inserted into the modified viral capsids for presentation of antigens to cells, e.g., for administration to a subject to generate an immune response to the exogenous amino acid sequences.

[0168] In some embodiments, the modified viral capsids herein can be targeted viral capsids comprising 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 insertion of an 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 insertion of a P1 peptide containing an RGD motif after amino acid positions 447, 534, 573, and 587 of the AAV2 capsid subunit]). Other positions within the AAV capsid subunit that tolerate insertion are known in the art (e.g., positions 449 and 588 described by Grifman et al., Molecular Therapy 3:964-975 (2001)).

[0169] For example, the viral capsids of the application can have a relatively low efficiency tropism for certain target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm, kidney, brain, stomach, intestine, skin, endothelial cells, and / or lung). Targeting sequences can be advantageously incorporated into these low transduction vectors, thereby conferring the desired tropism and optionally selective tropism for a particular tissue to the viral capsid. AAV capsid proteins, capsids, and vectors comprising targeting sequences are described, for example, in International Patent Publication WO 00 / 28004. As another example, one or more naturally occurring amino acids described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)) can be incorporated into the AAV capsid subunit of the application at orthogonal sites as a means to redirect the low transduction vector to a desired target tissue. These non-natural amino acids can be advantageously used to chemically attach a molecule of interest to the AAV capsid protein, including but not limited to: a glycan (mannose-dendritic cell targeting); an RGD, bombesin, or neuropeptide for targeted delivery to specific cancer cell types; an RNA aptamer or peptide selected from phage display that targets to a specific cell surface receptor (such as a growth factor receptor, integrin, etc.). Methods of chemically modifying amino acids are known in the art (see, e.g., Greg T. Hermanson, Bioconjugate Techniques, 1stEd., Academic Press, 1996). Bioconjugate Techniques

[0170] In some embodiments, the targeting sequence can be a viral capsid sequence that directs infection to a specific cell type (e.g., a parvovirus capsid sequence, an AAV capsid sequence, or any other viral capsid sequence). ​

[0171] 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 typically does not bind a HS receptor (e.g., AAV9) in order to impart heparin binding to the resulting mutant. In another non-limiting example, the erythrocyte glycosphingolipid receptor binding domain of the B19 capsid can be substituted into an AAV capsid protein of the application to target the viral capsid or viral vector comprising the viral capsid to erythroid cells.

[0172] In some embodiments, an exogenous targeting sequence for use herein can be any amino acid sequence that encodes a peptide that alters the tropism of a viral capsid or viral vector comprising a modified AAV capsid protein. In some embodiments, the targeting peptide or protein can be naturally occurring, or alternatively, wholly or partially synthetic. In some examples, targeting sequences can include ligands and other peptides that bind 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 factors I and II, etc.), cytokines, melanocyte hormones (e.g., alpha, beta, or gamma), neuropeptides and endorphins, etc., as well as 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, chicken egg white lysozyme, erythropoietin, gonadotropin-releasing hormone, cortistatin, beta-endorphin, leu- enkephalin, rimorphin, alpha-neo-endorphin, angiotensin, pneumolysin, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof, as described above. As yet a further alternative embodiment, binding domains from toxins (e.g., tetanus toxin or snake toxins, such as alpha-bungarotoxin, etc.) can be substituted into the capsid protein as a targeting sequence. In some embodiments, the AAV capsid protein can be modified by substitution of a "non-classical" import / export signal peptide (e.g., fibroblast growth factor-1 and -2, interleukin 1, HIV-1 Tat protein, herpes virus VP22 protein, etc.) into the AAV capsid protein as described by Cleves (1997). Peptide motifs that direct uptake by specific cells are also contemplated, such as the FVFLP (SEQ ID NO:41) peptide motif triggers uptake by hepatocytes. In some embodiments, a targeting sequence for use herein can be a peptide that can be used for chemical coupling to another molecule that targets entry into a cell (e.g., can comprise arginine and / or lysine residues that can be chemically coupled through their R groups). Current Biology 7:R318 (1997)) into the AAV capsid protein. Peptide motifs that direct uptake by specific cells are also contemplated, such as the FVFLP (SEQ ID NO:41) peptide motif triggers uptake by hepatocytes. In some embodiments, a targeting sequence for use herein can be a peptide that can be used for chemical coupling to another molecule that targets entry into a cell (e.g., can comprise arginine and / or lysine residues that can be chemically coupled through their R groups).

[0173] In some embodiments, the capsid protein variants, viral capsids, and / or AAV vectors (e.g., ccAVV) disclosed herein can have equivalent or enhanced transduction efficiency relative to the transduction efficiency of the capsid protein variant, viral capsid, and / or AAV serotype from which the vector is derived. In some embodiments, the capsid protein variants, viral capsids, and / or AAV vectors (e.g., ccAVV) disclosed herein can have reduced transduction efficiency relative to the transduction efficiency of the capsid protein variant, viral capsid, and / or AAV serotype from which the vector is derived. In some embodiments, the capsid protein variants, viral capsids, and / or AAV vectors (e.g., ccAVV) disclosed herein can have equivalent or enhanced tropism relative to the tropism of the capsid protein variant, viral capsid, and / or AAV serotype from which the vector is derived. In some embodiments, the capsid protein variants, viral capsids, and / or AAV vectors (e.g., ccAVV) disclosed herein can have altered or different tropism relative to the tropism of the capsid protein variant, viral capsid, and / or AAV serotype from which the vector is derived. In some embodiments, the capsid protein variants, viral capsids, and / or AAV vectors (e.g., ccAVV) disclosed herein can have or 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 elicit a diminished immune response relative to the immune response of the capsid protein variant, viral capsid, and / or AAV serotype from which the vector is derived. In some embodiments, the capsid protein variants, viral capsids, and / or AAV vectors (e.g., ccAVV) disclosed herein can be administered to a subject in multiple doses (e.g., about two doses, about three doses, about four doses, about 5 doses, about 10 doses, about 15 doses, about 20 doses, about 40 doses, a number of doses required to observe one or more desired responses) relative to the number of doses that can be administered using the capsid protein variant, viral capsid, and / or AAV serotype from which the vector is derived.

[0174] (A) Capsid and ccAAV engineering

[0175] In some embodiments, rational engineering and / or mutagenesis 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 generate AAV vectors that evade neutralizing antibodies. In some embodiments, the methods herein can be used to generate AAV vectors with improved gene transfer efficiency. In some embodiments, the methods herein can be used to generate AAV vectors with improved gene transfer efficiency in more than one mammalian species. In some embodiments, the methods herein can be used to generate AAV vectors that specifically target a cell or tissue of interest (e.g., kidney cells).

[0176] In some embodiments, the recombinant AAVs described herein have improved gene transfer efficiency in one or more mammalian species relative to a recombinant AAV having a capsid protein that is otherwise identical except that it lacks one or more amino acid substitutions. In some embodiments, the improved gene transfer efficiency occurs in one or more of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primates (Macaca, macaque), 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 ganglion, 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.

[0177] Aspects of the disclosure provide methods of producing the AAV vectors disclosed herein. In some examples, the method can comprise one or more of the following steps: a) identifying contact amino acid residues that form a three-dimensional antigenic footprint on an AAV capsid protein; b) generating a library of AAV capsid proteins comprising amino acid substitutions of the contact amino acid residues identified in (a); c) producing AAV particles comprising the capsid proteins from the library of AAV capsid proteins of (b); d) contacting the AAV particles of (c) with cells under conditions in which infection and replication can occur; e) selecting AAV particles that can complete at least one infectious cycle and replicate to a titer similar to a control AAV particle; f) contacting the AAV particles selected in (e) with neutralizing antibodies and cells under conditions in which infection and replication can occur; and g) selecting AAV particles that are not neutralized by the neutralizing antibodies of (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 variety of methods and protocols that are continually evolving that can be used to generate libraries of AAV capsid proteins (e.g., rational design, barcoding, directed evolution, computer discovery). Any method known in the art or to be discovered that is suitable for use herein can be used and / or optimized to generate a library of AAV capsid proteins according to the methods disclosed herein.

[0178] In some embodiments, generating a library of AAV capsid proteins comprising amino acid substitutions of the identified contact amino acid residues in the AAV capsid protein can generate a parent AAV capsid protein library. In some embodiments, the method of producing a ccAAV vector herein can comprise administering the parent AAV capsid protein library to a mammal. In some embodiments, administering the parent AAV capsid protein library to the mammal can be administering systemically to the mammal. In some embodiments, the parent AAV capsid protein library can be administered to a mammal having a Mus musculus (mouse), Sus scrofa (pig), Canis familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human) species. In some embodiments, the capsid proteins can be enriched by collecting from the cells and / or tissues of the mammal after administering the parent AAV capsid protein library. In some embodiments, the capsid proteins can be enriched by collecting from the cells and / or tissues of the mammal after administering the parent AAV capsid protein library, wherein the cells and / or tissues comprise spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, the 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 administering the parent AAV capsid protein library. In some embodiments, the capsid proteins collected from the mammal after administering the parent AAV capsid protein library can be used to generate another AAV capsid protein library called an evolved AAV capsid protein library.

[0179] In some embodiments, the evolved AAV capsid protein library can be administered to a mammal of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human) species, provided that the species is different from the species to which the parental AAV capsid protein library is administered. In some embodiments, the capsid proteins can be enriched by collecting from the cells and / or tissues of the mammal after administration of the evolved AAV capsid protein library. In some embodiments, the capsid proteins can be enriched by collecting from the cells and / or tissues of the mammal after administration of the evolved AAV capsid protein library, wherein the cells and / or tissues comprise spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, the capsid proteins can be collected and identified from the mammal after administration of the evolved AAV capsid protein library. In some embodiments, the capsid proteins can be collected and identified from the mammal after administration of the evolved AAV capsid protein library about 1 day to about 1 month (e.g., about 1 day, 5 days, 1 week, 2 weeks, 3 weeks, 1 month). In some embodiments, the capsid proteins collected and identified from the mammal after administration of the evolved AAV capsid protein library can be used to generate a further second evolved AAV capsid protein library. In some embodiments, the second evolved AAV capsid protein library can be administered to a mammal of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human) species, provided that the species is different from the species to which the first evolved AAV capsid protein library is administered. In some embodiments, the second evolved AAV capsid protein library can be administered to a mammal of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human) species, provided that the species is different from the species to which the first evolved AAV capsid protein library is administered, and the species is the same as the species to which the parental AAV capsid protein library is administered. In some embodiments, the second evolved AAV capsid protein library can be administered to a mammal of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human) species, provided that the species is different from the species to which the first evolved AAV capsid protein library is administered, and the species is different from the species to which the parental AAV capsid protein library is administered.

[0180] In some embodiments, each generation of the evolutionary library can be referred to as a“round” of the coevolution AAV capsid protein library. In some embodiments, the methods of coevolving an AAV capsid protein library herein can involve from about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) rounds. In some embodiments, each round as disclosed herein can be performed in a different species from the round it is performed. In some examples, the methods of coevolving an AAV capsid protein library herein can involve one round in a mouse, a second round in a pig, a third round in a mouse, a fourth round in a pig, etc. In some examples, the methods of coevolving an AAV capsid protein library herein can involve one round in a mouse, a second round in a non-human primate, a third round in a mouse, a fourth round in a non-human primate, etc. In some examples, the methods of coevolving an AAV capsid protein library herein can involve one round in a mouse, a second round in a pig, a third round in a non-human primate, a fourth round in a mouse, etc. In some examples, the methods of coevolving an AAV capsid protein library herein can involve one round in a pig, a second round in a mouse, a third round in a non-human primate (e.g., monkey), etc.

[0181] In some embodiments, the method of evolving a new adeno-associated virus strain comprises passaging an AAV library across multiple mammalian species, wherein the AAV library comprises a plurality of recombinant AAV vectors, wherein each recombinant AAV vector comprises 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 located in a region corresponding to amino acids 452-458 of SEQ ID NO: 1 or 586-592 of SEQ ID NO: 1, or the mutations are found in both regions corresponding to amino acids 452-458 and 586-592 of SEQ ID NO: 1.

[0182] In some embodiments, the method of evolving new AAV strains comprises administering a first AAV library to a first mammalian species. Then, AAV of the first AAV library present in one or more target tissues from the first mammalian species can 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 mammalian species and the second mammalian species are different. Then, AAV of the second AAV library present in one or more target tissues from the second mammalian species can be sequenced. In some embodiments, 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 lupus familiaris (dog), non-human primates (Macaca, Rhesus macaque), or 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 the group consisting of spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0183] (B) AAV vectors

[0184] In certain embodiments, the disclosure provides AAV vectors 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 carries a heterologous molecule. A “viral vector” is a vector that comprises one or more polynucleotide regions encoding or comprising a polypeptide or a multi-polypeptide of interest, a polynucleotide encoding a polypeptide or a multi-polypeptide, or a regulatory nucleic acid. Viral vectors of the present invention can be produced recombinantly using methods known in the art. These techniques are Molecular Cloning: A Laboratory Manual、 second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology , Humana Press; and Cell Biology: A Laboratory Notebook (J. E. Cellis,ed., 1989) Academic Press.

[0185] 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 (e.g., AAV) by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replacing it with a non-native nucleic acid such as a heterologous polynucleotide sequence (e.g., a coding sequence for a transgene of interest). Typically, for AAV vectors, 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 are replaced with non-native sequences (such as a heterologous polynucleotide sequence between the retained ITRs). The vector genomes disclosed herein can encompass a backbone element derived from an 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 genomes disclosed herein can further comprise regulatory sequences that modulate expression and / or secretion of the encoded protein. Examples include, but are not limited to, enhancers, polyadenylation signal sites, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTDs), microRNA target sites, or combinations thereof.

[0186] In some examples, the vector genomes described herein can be single-stranded. In other embodiments, the vector genomes disclosed herein can be double-stranded. For example, the vector genomes described herein can be self-complementary AAV vector genomes that are capable of containing double-stranded portions therein.

[0187] (1) AAV backbone elements

[0188] In some embodiments, the vector genomes disclosed herein can have one or more AAV genome-derived backbone elements, which refers to the minimal AAV genome elements required for the biological activity of an AAV vector. For example, the AAV genome-derived backbone elements can include a packaging site for the vector to be assembled into an AAV viral particle, one or more of the capsid protein variants disclosed herein, elements required for replication of the vector, and / or expression of a transgene coding sequence contained therein in a host cell.

[0189] In some examples, the vector genome backbone disclosed herein can comprise at least one inverted terminal repeat sequence (ITR). In some examples, the vector genome backbone herein can comprise two ITR sequences. In some examples, one ITR sequence can be 5' of the polynucleotide sequence encoding a transgene. In some examples, one ITR sequence can be 3' of the polynucleotide sequence encoding a transgene. In some examples, the polynucleotide sequence encoding a transgene herein can be flanked on either side by an ITR sequence. Thus, in some embodiments, the vector genome comprises a transgene located between a first ITR and a second ITR.

[0190] In some embodiments, the vector genomes herein can include sequences or components derived from at least one different AAV serotype. In some examples, the AAV vector genome backbones disclosed herein can comprise at least the ITR sequences from one different AAV serotype. In some examples, the AAV vector genome backbones disclosed herein can comprise at least the ITR sequences from one different human AAV serotype. Such human AAV can be derived from any of the known serotypes, for example from any of serotypes 1-11. In some examples, the AAV serotypes used herein are tropic for the central nervous system (CNS), cardiac tissue, skeletal muscle, and / or liver tissue. In some examples, the AAV vector genome backbones disclosed herein can have the ITR sequences of serotype AAV9.

[0191] In some embodiments, the AAV vectors herein can be pseudotyped AAV vectors (i.e., comprising sequences or components derived from at least two different AAV serotypes). In some embodiments, the pseudotyped AAV vectors herein can include an AAV genome backbone derived from one AAV serotype and capsid proteins at least partially derived from a different AAV serotype. In some examples, the pseudotyped AAV vectors herein can have an AAV2 vector genome backbone and capsid proteins derived from an AAV serotype that is tropic for cardiac tissue, such as AAV1, AAV2, AAV4, AAV5, AAV8, or AAV9.

[0192] To analyze the success of viral vector-mediated gene transfer, it can be important to be able to monitor the distribution of the vector and the effectiveness of vector-mediated gene expression. This can be accomplished by subcloning a reporter gene into the vector genome backbone. In some examples, the AAV vector genome backbones 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), red fluorescent protein (RFP), E. coli beta-galactosidase (LacZ), and various forms of luciferase LacZ ) and various forms of luciferase (Luc) In some examples, the AAV vector backbones disclosed herein can contain GFP.

[0193] The vector constructs disclosed herein can be made using known techniques. (See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Sambrook and Russell, Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2006); Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, Inc., Hoboken, N.J. (1999); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., Hoboken, N.J. (2002). Current Protocols in Molecular BiologyAusubel, F. et al., eds, Wiley and Sons, New York 1995). The fragment length can be chosen such that the recombinant genome does not exceed the packaging capacity of the AAV particle. If desired, "stuffer" DNA sequences can be added to the construct to maintain the standard AAV genome size for comparison purposes. Such fragments can be derived from such non-viral sources, e.g., lacZ, or other genes known and available to those skilled in the art.

[0194] (2) Self-complementary AAV viral vectors

[0195] In some embodiments, the AAV vectors disclosed herein can be self-complementary AAV (scAAV) vectors. Self-complementary AAV (scAAV) vectors contain complementary sequences that are able to self-anneal (fold back on itself to form a double-stranded genome) upon entry into an infected cell, thus avoiding the need to use the cell's DNA replication machinery to convert a single-stranded DNA vector. An AAV with a self-complementary genome can rapidly form a double-stranded DNA molecule through its partially complementary sequences (e.g., the coding and non-coding strands of a complementary transgene coding sequence).

[0196] In some embodiments, the scAAV viral vectors disclosed herein can comprise a first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence, which can form 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; e.g., to form a hairpin DNA structure. In some examples, the dimeric structure of the scAAV vector upon entry into a cell can be stabilized by mutation or deletion of one of the two terminal resolution sites (trs). As the trs is a Rep-binding site contained within each ITR, such mutation or deletion of the trs can prevent the AAV Rep protein from cleaving the dimeric structure of the scAAV vector to form monomers. In some embodiments, the scAAV viral vectors disclosed herein can comprise a truncated 5' inverted terminal repeat sequence (ITR), a truncated 3' ITR, or both. In some examples, the scAAV viral vectors disclosed herein can comprise a truncated 3' ITR, wherein the D region or a portion thereof (e.g., the terminal resolution sequence therein) can be deleted. Such truncated 3' ITR can be located between the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence described above.

[0197] (3) Promoters

[0198] In some embodiments, the AAV vectors disclosed herein can further comprise other elements necessary for expression, such as at least one suitable promoter that controls expression of the transgene coding sequence upon infection of a suitable cell. Suitable promoters for use herein include, in addition to AAV promoters, for example, cytomegalovirus (CMV) promoters or chicken beta actin / cytomegalovirus hybrid promoters (CAG), endothelial cell-specific promoters (such as VE-cadherin promoters), as well as steroid and metallothionein promoters. In some embodiments, the promoter used in the vectors disclosed herein can be a CAG promoter.

[0199] In some embodiments, the AAV vectors disclosed herein can further comprise other elements necessary for expression, such as at least one suitable promoter that controls expression of the transgene coding sequence upon infection of a suitable cell. Suitable promoters for use herein include, in addition to AAV promoters, for example, cytomegalovirus (CMV) promoters or chicken beta actin / cytomegalovirus hybrid promoters (CAG), endothelial cell-specific promoters (such as VE-cadherin promoters), as well as steroid and metallothionein promoters. In some embodiments, the promoter used in the vectors disclosed herein can be a CAG promoter.

[0200] In some embodiments, the transgene coding sequence according to the present application comprises a tissue-specific promoter functionally linked to the transgene coding sequence to be expressed. Thus, the specificity of the vectors according to the present disclosure to a tissue (e.g., brain, heart, muscle, liver) can be further increased. In some examples, the vectors disclosed herein can have a tissue-specific promoter that is at least about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more active in a particular tissue than in a tissue that is not the particular tissue. In some examples, the tissue-specific promoter herein is a human tissue-specific promoter. In some examples, the expression cassette can further include an enhancer element for increasing the expression level of the exogenous protein to be expressed. In addition, the expression cassette can further comprise a polyadenylation sequence, such as an SV40 polyadenylation sequence or a polyadenylation sequence of bovine growth hormone.

[0201] (4) Other regulatory elements for gene expression

[0202] In some embodiments, the AAV vectors disclosed herein can include one or more conventional control elements operably linked to the transgene coding sequence in a manner that permits their transcription, translation, and / or expression in a cell transfected with a plasmid vector or infected with a virus produced by the application. As used herein, "operably linked" sequences can include 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 can further include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A large number of expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used herein.

[0203] In some embodiments, the AAV vectors disclosed herein can include a modified capsid, including a protein or peptide of non-viral origin or structural modification to alter the tropism of the vector. For example, the capsid can include a ligand for a particular receptor, or a receptor for a particular ligand, to target the vector to cell types expressing the receptor or ligand, respectively.

[0204] (C) Serotypes of AAV viral particles

[0205] In some embodiments, the AAV vectors disclosed herein can be made from or derived from AAV of various serotypes. The term "serotype" is a distinction with respect to AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on a lack of cross-reactivity between antibodies and AAV as compared to other AAV. 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 particular AAV is generated in rabbits or other suitable animal models using an adeno-associated virus. In this assay, sera generated against a particular AAV is then tested for the ability to neutralize the same (homologous) or a different (heterologous) AAV. The dilution at which 50% neutralization is achieved is considered the neutralizing antibody titer. If the quotient of the heterologous titer divided by the homologous titer for two AAVs is less than 16 in the inverse, then the two vectors are considered to be the same serotype. Conversely, if the ratio of the heterologous titer to the homologous titer is 16 or greater, then the two AAVs are considered to be different serotypes.

[0206] In some embodiments, the AAV vectors herein can be AAV of at least two serotypes or mixed with other types of viruses to produce chimeric (e.g., pseudotyped) AAV viruses. In some embodiments, the AAV vectors herein can be human serotype AAV vectors. Such human AAV can be derived from any of the known serotypes, for example, from any of serotypes 1-11.

[0207] (D) Methods of making AAV particles

[0208] In some embodiments, the AAV vector genomes described herein can be packaged into viral particles that can be used to deliver the genome for expression of the transgene coding sequence in target cells. In some embodiments, the AAV vector genomes disclosed herein can be packaged into particles by transient transfection, using a producer cell line, combining viral features into Ad-AAV hybrids, using a herpes virus system, or using a baculovirus in insect cells.

[0209] Methods of producing packaging cells for use herein can include producing a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking 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) is integrated into the genome of a cell. The AAV genome is introduced into the bacterial plasmid by methods such as GC tailing, addition of synthetic linkers containing restriction endonuclease cleavage sites, or by direct blunt-end ligation. The packaging cell line is then infected with a helper virus (such as an adenovirus). The advantage of this method is that the cells are selectable and amenable to large-scale production of rAAV. Examples of suitable methods herein use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0210] (E) Characteristics of AAV vectors and AAV particles

[0211] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can have one or more improvements compared to a naturally isolated AAV vector. As used herein, “naturally isolated AAV vector” refers to a vector that does not comprise one or more of the capsid protein variants disclosed herein. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can have increased gene transfer efficiency in cells compared to a naturally isolated AAV vector. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can have 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) increased gene transfer efficiency in cells compared to a naturally isolated AAV vector.

[0212] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can 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 can have increased gene transfer efficiency in cells and / or tissues of one or more of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primates (Macaca, Macaca), or Homo sapiens (human), and any combination thereof. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can have increased gene transfer efficiency in cells and / or tissues of mammalian spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0213] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can have a higher vector titer compared to a naturally isolated AAV vector. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can have 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 vector titer compared to a naturally isolated AAV vector.

[0214] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be less susceptible to antibody-mediated neutralization compared to naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be less susceptible to antibody-mediated neutralization by 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 naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be less susceptible to antibody-mediated neutralization 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 compared to naturally isolated AAV vectors.

[0215] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can produce lower levels of anti-AAV antibodies after at least one administration to a subject compared to naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can produce 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 anti-AAV antibodies after at least one administration to a subject compared to naturally isolated AAV vectors. In some embodiments, gene therapy comprising the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be administered to a subject herein about 2 times to about 10 times (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10 times) without becoming susceptible to antibody-mediated neutralization.

[0216] 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 a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, or a non-human primate (e.g., a monkey, a chimpanzee, a baboon, a gorilla). In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be expressed in any cell or tissue type of a human, a mouse, a dog, and a non-human primate.

[0217] III. Pharmaceutical Compositions

[0218] 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 a pharmaceutical composition. In some examples, the pharmaceutical compositions herein can further comprise a pharmaceutically acceptable carrier, diluent, or excipient. Any pharmaceutical composition used in the methods of the present application can comprise a pharmaceutically acceptable carrier, excipient, or stabilizer in lyophilized form or in aqueous solution.

[0219] The carrier in the pharmaceutical composition must be "acceptable" in the sense of being compatible with the active ingredients of the composition and preferably capable of stabilizing the active ingredients and not deleterious to the subject to be treated. For example, "pharmaceutically acceptable" can refer to molecular entities and other ingredients of the composition that are physiologically tolerable and do not typically produce adverse reactions when administered to a mammal (e.g., a human). In some examples, a "pharmaceutically acceptable" carrier used in the pharmaceutical compositions disclosed herein can be those approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0220] Pharmaceutically acceptable carriers (including buffers) are well known in the art and can include phosphate, citrate, 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; hydrophilic 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 Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0221] In some embodiments, the pharmaceutical composition or formulation is for parenteral administration, e.g., intravenous, intracerebroventricular injection, intracisternal injection, intraparenchymal injection, or a combination thereof. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of

[0222] Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The aqueous solutions can be suitably buffered (preferably pH 3-9) as desired. Preparation of appropriate parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques known to those with ordinary skill in the art.

[0223] Pharmaceutical compositions for in vivo administration should be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Sterile injectable solutions are prepared by incorporating the active compound (e.g., AAV vector (e.g., ccAAV), viral capsid, and / or AAV viral particle) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0224] The pharmaceutical compositions disclosed herein can also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, 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 dextrins; 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, pluronics, or polyethylene glycol.

[0225] IV. Methods of Use

[0226] 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 disorder. Accordingly, in some aspects, the present disclosure provides methods of reducing one or more symptoms and / or treating a disease or disorder in a subject in need of treatment by the compositions disclosed herein and pharmaceutical compositions comprising such compositions. In some embodiments, the subject of the methods herein can be a human subject. In some embodiments, the subject can be a subject that has not been previously exposed to wild-type AAV or a recombinant (rAAV) vector. In some embodiments, the subject can be a subject that has not been previously administered a rAAV vector. In some embodiments, the subject is a subject that has been previously administered a rAAV vector, e.g., a rAAV vector described herein. Subjects that have been exposed or administered AAV or rAAV can be identified using methods known in the art, e.g., by PCR detection of viral DNA or by measuring antibody titers against AAV or rAAV (capsid or transgene). In some embodiments, the subject can be a subject that has not been administered enzyme replacement therapy (e.g., by administration of an enzyme protein). Subjects that have been administered enzyme replacement therapy can be identified using methods known in the art, e.g., by measuring antibody titers against the enzyme. However, in some embodiments, the subject has been previously treated with enzyme replacement therapy. In some embodiments, the subject is a subject that has undergone one or more methods of clearing neutralizing antibodies (NAbs) (e.g., plasmapheresis, immunosuppression, enzymatic degradation). In some embodiments, a subject suitable for the methods of use herein can not need to clear neutralizing antibodies (NAbs) prior to administration of any of the compositions described herein (e.g., AAV vectors (e.g., ccAAV), viral capsids, and / or AAV viral particles).

[0227] In some embodiments, the subject has or is suspected of having a disease that can be treated with gene therapy. Illustrative diseases or conditions that can be treated using the methods disclosed herein can include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulator) and other diseases of the lung, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (beta-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; enkephalinase), multiple sclerosis (beta-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophins), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAi including RNAi against VEGF or multi-drug resistance gene products, mir-26a [e.g., for hepatocellular carcinoma]), diabetes (insulin), muscular dystrophy including Duchenne (anti-dystrophin, micro-dystrophin, insulin-like growth factor I, sarcoglycans [e.g., alpha, beta, gamma], RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides such as IKB dominant mutants, sarcospan, utrophin, mini-utrophin, antisense or RNAi against splice junctions in the anti-dystrophin gene to induce exon skipping [see, e.g., WO / 2003 / 095647], antisense against U7 snRNA to induce exon skipping [see, e.g., WO / 2006 / 021724], or antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker, Gaucher disease (glucocerebrosidase), Hurler disease (alpha-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [-alpha galactosidase] and Pompe disease [lysosomal acid alpha glucosidase]) and other metabolic disorders, congenital lung emphysema (alpha 1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain keto acid 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 angiogenic inhibitors to treat / prevent retinal disorders, e.g., in type I diabetes), diseases of solid organs such as brain (including Parkinson's disease [GDNF], astrocytoma [endostatin, vasohibin and / or RNAi against VEGF], glioblastoma [endostatin, vasohibin and / or RNAi against VEGF]), liver, kidney, heart including congestive heart failure or peripheral arterial disease (PAD) (e.g., by delivering protein phosphatase inhibitor I (I-1) and fragments thereof (e.g., I1C), serca2a, zinc finger protein that regulates the gene for phospholamban, Barkct, P2-adrenergic receptor, p2-adrenergic receptor kinase (BARK), phosphatidylinositol-3 kinase (PI3 kinase), S100A1, albumin, adenylyl cyclase type 6, molecules that affect G protein-coupled receptor kinase type 2 knockdown such as truncated constitutively active bARKct; calsarcin, RNAi against phospholamban; phospholamban inhibiting or dominant negative molecules such as phospholamban S16E, etc.), arthritis (insulin-like growth factor), arthritic conditions (insulin-like growth factors 1 and / or 2), intimal hyperplasia (e.g., by delivering enos, inos), improving survival of heart transplants (superoxide dismutase), AIDS (soluble CD4), muscle atrophy (insulin-like growth factor I), renal failure (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNF alpha soluble receptor), hepatitis (alpha-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, spinocerebellar ataxias including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc.

[0228] In some embodiments, the AAV vectors, compositions, and methods described herein can be used to treat a kidney disease or a kidney disorder, such as Alport syndrome, benign familial hematuria, polycystic kidney disease (e.g., type 1 or type 2), vascular nephropathy, nephrogenic diabetes insipidus, familial hypocalciuric hypercalcemia, nephrocalcinosis, hypophosphatemic rickets, Fabry disease, nephroblastoma, or steroid-resistant nephrotic syndrome.

[0229] To perform the methods disclosed herein, an effective amount of a composition (e.g., an AAV vector (e.g., ccAAV), a viral capsid, and / or an AAV viral particle) or a pharmaceutical composition comprising such a composition can be administered to a subject in need of treatment in a suitable amount disclosed herein via 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).

[0230] In certain embodiments, the present disclosure also provides methods of introducing one or more AAV vectors (e.g., ccAAV) into a cell, comprising contacting the cell with a composition disclosed herein. In some embodiments, the methods herein can comprise delivering one or more AAV vectors (e.g., ccAAV) herein to a cell, 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 vectors (e.g., ccAAV) herein can deliver one or more heterologous molecules to the cell. In accordance with these embodiments, the AAV vectors (e.g., ccAAV) herein can deliver one or more therapeutic heterologous molecules to the cell. In some examples, the one or more therapeutic heterologous molecules delivered to the cell using the methods herein can be a therapeutic protein, a therapeutic DNA, and / or a 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 can be an antisense oligonucleotide, an siRNA, an shRNA, an mRNA, a DNA oligonucleotide, and the like.

[0231] In certain embodiments, the present disclosure also provides methods of introducing an AAV vector (e.g., ccAAV) into CNS tissue, cardiac tissue, kidney tissue, liver tissue, skeletal muscle tissue, or any combination thereof, comprising contacting the cell with a viral vector and / or a 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 tropism for CNS tissue, cardiac tissue, kidney tissue, liver tissue, skeletal muscle tissue, or any combination thereof.

[0232] In some embodiments, the method of administering to a tissue at least one AAV vector (e.g., ccAAV), viral capsid, and / or AAV viral particle having one or more nucleic acid molecules herein substantially modulates 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 encoded product of the transgene) prior to administration of an AAV vector (e.g., ccAAV) herein. As used herein, “substantially modulates expression” refers to a change in expression (e.g., increase in expression, decrease in expression) of at least 1-fold compared to baseline. In some embodiments, the method of administering to a tissue at least one AAV particle or AAV vector (e.g., ccAAV) having one or more AAV capsid protein variants disclosed herein modulates expression of at least one protein and / or gene compared to baseline 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). In some embodiments, the method of administering to a tissue at least one AAV particle or AAV vector (e.g., ccAAV) having one or more AAV capsid protein variants disclosed herein modulates expression of at least one protein and / or gene compared to baseline 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) 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.

[0233] In any of the methods disclosed herein, an effective amount of a composition described herein (e.g., AAV vector, viral capsid, AAV particle, AAV genome, ccAAV) can be administered to a subject in need thereof to reduce one or more symptoms associated with a disease and / or disorder. As used herein, “effective amount” refers to a dosage of a disclosed composition sufficient to impart a therapeutic effect on a subject having a disease and / or disorder. In some embodiments, an effective amount can be an amount that reduces at least one symptom of a disease or disorder in a subject.

[0234] In some embodiments, the method of administering at least one AAV vector as disclosed herein (e.g., ccAAV) can have increased gene transfer efficiency in a cell compared to a naturally isolated AAV vector. In some embodiments, the method of administering at least one AAV vector as disclosed herein can have 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) increased gene transfer efficiency in a cell compared to a naturally isolated AAV vector. In some embodiments, the method of administering at least one AAV vector as disclosed herein can have increased gene transfer efficiency in a tissue compared to a naturally isolated AAV vector. In some embodiments, the method of administering at least one AAV vector as disclosed herein can have 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) increased gene transfer efficiency in a tissue compared to a naturally isolated AAV vector. In some embodiments, the method of administering at least one AAV vector as disclosed herein can have increased gene transfer efficiency in a subject compared to a naturally isolated AAV vector. In some embodiments, the method of administering at least one AAV vector as disclosed herein can have 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) increased gene transfer efficiency in a subject compared to a naturally isolated AAV vector.

[0235] In some embodiments, the methods herein can comprise administering to the subject at least one AAV vector (e.g., ccAAV) at least once. In some embodiments, the methods herein can comprise administering to the subject at least one AAV particle and / or at least one AAV vector more than once. In some embodiments, the methods herein can comprise administering to the subject at least one AAV vector herein 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 can comprise administering to the subject at least one AAV vector herein at least two times, at least 3 times, at least 4 times, or at least 5 times. In some embodiments, the methods herein can comprise administering to the subject at least one AAV vector herein once a day, once every other day, once a week, once every two weeks, once every three weeks, once a month, once every other month, once every three months, once every four months, once a year, or twice a year. In some embodiments, the methods herein can comprise administering to the subject at least one AAV vector herein as many times as necessary to observe a desired response. In some examples, the desired response can be a reduction in at least one symptom of a disease and / or disorder in the subject after administration of a dose of an AAV vector herein compared to prior to administration of the AAV vector. Those skilled in the art will appreciate that the dosing regimen can be optimized according to the disease / disorder, the severity of the disease / disorder, the characteristics of the subject (e.g., age, sex, weight), and the like.

[0236] In some embodiments, the AAV vectors herein (e.g., ccAAV) can be used to deliver a cre-recombinase. In some embodiments, the AAV vectors herein (e.g., ccAAV) can be used to deliver a cre-recombinase to cause conditional activation, conditional inactivation, activation, inactivation, or any combination thereof, of one or more genes in a cell, tissue, and / or subject. According to some of these embodiments, the AAV vectors herein (e.g., ccAAV) deliver the cre-recombinase to one or more specific cell and / or tissue types.

[0237] In some embodiments, the AAV vectors herein (e.g., ccAAV) can be used to deliver a CRISPR-Cas system. A “CRISPR / Cas9” system or “CRISPR / Cas9-mediated gene editing” refers to a Type II CRISPR / Cas system that has been modified for genome editing / engineering. It typically consists of a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). “Guide RNA (gRNA)” is used interchangeably herein with “short guide RNA (sgRNA)” or “single guide RNA (sgRNA).” sgRNA is a short synthetic RNA that consists of a “scaffold” sequence necessary for Cas9 binding and a user-defined ~20 nucleic acid “spacer” or “targeting” sequence that defines the genomic target to be modified. The genomic target of Cas9 can be changed by changing the targeting sequence present in the sgRNA.

[0238] In some embodiments, the AAV vector comprises a vector genome, wherein 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.

[0239] V. Kits

[0240] The present disclosure also provides kits for making any of the compositions (e.g., AAV vectors, AAV particles, AAV genomes, viral capsids, ccAAV) as described herein and kits having one or more therapeutic uses as described herein. Kits for use as described herein can include one or more containers further comprising a composition (e.g., AAV vectors, AAV particles, AAV genomes, viral capsids, ccAAV) as described herein formulated in a pharmaceutical composition.

[0241] In some embodiments, the kits can additionally comprise instructions for using the compositions (e.g., AAV vectors, AAV particles, AAV genomes, viral capsids, ccAAV) in any of the methods described herein. The included instructions can include a description of administering the compositions or pharmaceutical compositions comprising such compositions to a subject to achieve the intended activity in the subject. The kit can further include instructions for selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. The instructions related to the use of the compositions described herein generally include information regarding the dosage, dosing regimen, and route of administration for the intended treatment.

[0242] The container can be a unit-dose, bulk package (e.g., a multi-dose package), or a subunit-dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used for treating, delaying the onset of, and / or lessening a disease or condition of a subject.

[0243] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with particular devices, such as inhalers, nasal administration devices, or infusion devices. The kits can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port.

[0244] The kits optionally can provide additional components such as buffers and explanatory information. Typically, the kits include a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides articles of manufacture comprising the contents of the kits described above. DETAILED DESCRIPTION

[0245] Examples

[0246] While the application has been described with reference to the currently preferred embodiments, those skilled in the art will understand that various changes can be made and equivalents substituted for elements thereof without departing from the true spirit and scope of the application. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process steps or steps thereof, to the objective, spirit and scope of the present disclosure. All such modifications will be within the scope of the application.

[0247] Example 1. Cross-species evolution of AAV capsids

[0248] A method to generate co-evolved AAV capsid protein variants is as follows. The first step involves the identification of conformational 3D antigenic epitopes on the surface of AAV9 capsids using cryo-electron microscopy. Then, AAV9 libraries are engineered by saturation mutagenesis of the amino acid residues identified within the surface loops. Specifically, amino acid residues within variable region IV (452-NGSGQNQ-458; SEQ ID NO: 38) and within variable region VIII (586-SAQAQAQ-592; SEQ ID NO: 39) are selected for saturation mutagenesis and generate two different AAV libraries - a variable region IV (VR4) AAV parental library and a variable region VIII (VR8) parental library. Degenerate primers are used to mutagenize the selected residues within the antigenic motifs, where each codon is replaced with the nucleotides NNK, and the gene fragments are assembled together by Gibson assembly (a method based on sequence overlap). Specifically, to generate AAV VR4 (variable region IV) and VR8 (variable region VIII) libraries, oligonucleotides containing 21-mers (NNRNNRNNRNNRNNRNNRNNR; SEQ ID NO: 124) and homology arms to the AAV9 Cap gene are synthesized by integrated DNA technology, where “N” corresponds to any nucleotide (A, T, G, C) and “R” corresponds to G or C to prevent premature stop codons in the capsid library.

[0249] The resulting degenerate library containing mutated antigenic motifs is cloned into the capsid-encoding gene of the wild-type AAV genome to replace the DNA sequence encoding the original Cap, generating a plasmid library. Specifically, the plasmid contains genes encoding AAV2 Rep and AAV9 Cap flanked by AAV2 ITRs, where the amino acid mutations in AAV9 Cap are changed to stop codons to reduce wild-type AAV9 plasmid contamination.

[0250] The VR4 and VR8 parental plasmid libraries are then transfected into a HEK293 producer cell line with adenovirus helper plasmids to generate AAV VR4 capsid and AAV VR8 capsid parental libraries. Briefly, HEK293 cells are transfected with polyethylenimine at 70-80% confluency with an equimolar ratio of pTR-AAV9-library and adenovirus helper plasmid pXX680. HuH7 (human hepatocellular carcinoma) cells are cultured to ~75% confluency and infected with AAV9 library at 5,000 viral genomes / cell overnight. The next day, the media is replaced with media containing Ad5 at a multiplicity of infection (MOI) of 0.5. At 50-75% cytopathic effect, the supernatant is collected and incubated at 55°C for 30 minutes to inactivate Ad5. DNase I-resistant viral genomes in the media are quantified and used as inoculum for the subsequent round of infection.

[0251] 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 in a more efficient manner than naturally occurring AAV9, the AAV library prepared as described above was subjected to multiple rounds or “cycles” of infection in three different mammalian species. In the first cycle, the AAV VR4 capsid parental library or the AAV VR8 capsid parental library prepared as described above was injected intravenously (i.v.) at about 3 x 1010vg / kg (viral genomes per kilogram) into 4-week-old piglets. Six days post-injection, the piglets were sacrificed and viral DNA was amplified from genomic DNA extracted from various brain regions (cerebellum, frontal lobe, temporal lobe, parietal lobe, occipital lobe cortex, hippocampus, thalamus, and midbrain) using oligonucleotides targeting the VR4 or VR8 flanking DNA sequences by PCR to amplify the AAV library sequences. Briefly, to amplify the evolved AAV library from this first cycle, DNase I-resistant viral genomes were isolated from the harvested pig brain tissue and amplified by Q5 polymerase for 10-18 cycles using primers for variable regions IV and VIII 5’- CCCTACACGACGCTCTTCCGATCTNNNNNGTACCTGTACTACTTGTCTCG-3’ (SEQ ID NO: 42) and 5’- GACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNAGACCATACCGGGTAAG-3’ (SEQ ID NO: 43). 13 -5 x 1010 13 vg / kg (viral genomes per kilogram) into 4-week-old piglets. Six days post-injection, the piglets were sacrificed and viral DNA was amplified from genomic DNA extracted from various brain regions (cerebellum, frontal lobe, temporal lobe, parietal lobe, occipital lobe cortex, hippocampus, thalamus, and midbrain) using oligonucleotides targeting the VR4 or VR8 flanking DNA sequences by PCR to amplify the AAV library sequences. Briefly, to amplify the evolved AAV library from this first cycle, DNase I-resistant viral genomes were isolated from the harvested pig brain tissue and amplified by Q5 polymerase for 10-18 cycles using primers for variable regions IV and VIII 5’- CCCTACACGACGCTCTTCCGATCTNNNNNGTACCTGTACTACTTGTCTCG-3’ (SEQ ID NO: 42) and 5’- GACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNAGACCATACCGGGTAAG-3’ (SEQ ID NO: 43).

[0252] Illumina MiSeq sequencing adaptors for multiplexed detection were added in the second round of PCR using Q5 polymerase and primers. After each round of PCR, the products were purified using the PureLink PCR Micro Kit (Invitrogen). The quality of the amplicons was verified using a Bioanalyzer (Agilent) and the concentration was quantified using a Qubit spectrometer (Invitrogen). The PCR amplicons containing the VR4 or VR8 library determined by Sanger sequencing were then pooled together and used to generate the next library preparation.

[0253] The resulting amplicons were then cloned back into vectors to generate the evolved plasmid libraries using the same methods as for the generation of the parental plasmid libraries, except that the amplicons were assembled using a multiplex overlap extension PCR, instead of Gibson assembly. The VR4 and VR8 parental plasmid libraries were then transfected into HEK293 producer cell lines with adenovirus helper plasmids, resulting in AAV VR4 capsid and AAV VR8 capsid evolved libraries using the same methods described above. Viral genomes resistant to DNase I in culture media were quantified and used as inoculum for the next round of infection.

[0254] In the second round of evolution in pigs, the AAV VR4 capsid parental library or the AAV VR8 capsid evolved library prepared as described above was injected intravenously (i.v.) at about 3x10 13 -5x10 13 vg / kg into 8-week-old C57 / B6 mice. Mice were sacrificed 6 days post-injection and viral DNA was amplified from genomic DNA extracted from various brain regions (cerebellum, frontal lobe, temporal lobe, parietal lobe, occipital lobe cortex, hippocampus, thalamus and midbrain) using oligonucleotides targeting the VR4 or VR8 flanking DNA sequences by PCR to amplify AAV library sequences as described above. The resulting amplicons were then cloned back into vectors to generate another evolved plasmid library using the same methods as for the generation of the first evolved plasmid library described above. Viral genomes in culture media were this time quantified and used as inoculum for the third round of evolution.

[0255] After evolution in pigs and mice, the evolved VR4 and VR8 libraries were injected intravenously (i.v.) at about 1x10 13 -3x10 13 vg / kg into 2-year-old non-human primates (NHPs). Viral DNA was amplified from genomic DNA extracted from various brain regions in NHPs as described above. Amplified viral DNA was subjected to high-throughput sequencing using the lllumina MiSeq platform and the resulting data was analyzed as follows.

[0256] Quality control checks were performed on the multiplexed reads using FastQC (v.0.11.5) and sequences of poor quality were not flagged and analyzed using similar methods as Tse et al., 2012, PLoS One 7(7): e39832. PNASThe methods described in Wang, et al. (2017) Nat Biotechnol. 2017 Jun 13; 114(24): E4812-E4821 were analyzed by custom Perl scripts, the disclosure of which is incorporated by reference in its entirety. Briefly, the target mutagenized region in the raw sequencing files was probed and the frequency of different nucleotide sequences in that region was counted and binned for each library. Nucleotide sequences were also translated and similar counts and binning was performed on these amino acid sequences. The frequency of amino acid sequences in the libraries was then plotted in the R graphics package v3.5.2. A second Perl script was used to calculate the amino acid representation at each position in each library, taking into account the contribution of each mutant in the library.

[0257] These libraries were subjected to multiple rounds of evolution between three species (pig, mouse, and NHP (i.e., monkey)), resulting in several AAV9 capsid variants. Sequencing was performed on AAV9 capsid variants from the cross-species in vivo screen with the highest frequency. Bubble plots show the enrichment of library diversity, directed evolution, and neoantigen footprints in the VR8 region and VR4 region between the parental libraries Figure 1A ) and the evolved libraries after three cycles between three different species Figure 1B ). Substitutions present in these AAVs in region IV (452-NGSGQNQ-458; SEQ ID NO: 38) or region VIII (586-SAQAQAQ-592; SEQ ID NO: 39) are shown in Table 5.

[0258] Table 5

[0259]

[0260] Example 2. In vivo characterization of recombinant AAV in mice

[0261] 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 AAVs containing these capsid proteins or native AAV9 and packaging a fluorescent transgene were generated. Briefly, the recombinant capsid proteins generated were packaged as vectors for 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 with polyethylenimine at 70-80% confluency using a triple-plasmid transfection protocol. Using this method, recombinant vectors were produced that packaged single-stranded genomes encoding either green fluorescent protein driven by a hybrid chicken beta-actin promoter (CBh-eGFP), cherry (red) fluorescent protein driven by a hybrid chicken beta-actin promoter (CBh-mCherry), or self-complementary AAV9 driven by either CBh-eGFP or CBh-mCherry. See generally Figures 29A-29B . Subsequent steps including harvesting of recombinant AAV vectors and downstream purification were performed. Briefly, vector purification was performed using an iodaxinol gradient ultracentrifugation protocol, and vivaspin2 100 kDa molecular weight cut-off (MWCO) centrifugal columns (F-2731-100 Bioexpress) were used for buffer exchange and concentration. Recombinant AAV vector titers were determined by quantitative PCR with primers amplifying the AAV2 inverted terminal repeat region (ITRs) 5'- AACATGCTACGCAGAGAGGGAGTGG-3' (SEQ ID NO: 44) and 5'- CATGAGACAAGGAACCCCTAGTGATGGAG-3' (SEQ ID NO: 45).

[0262] C57 / BL mice were injected intravenously with self-complementary AAV9 or one of the ccAAV vectors at a dose of 5 x 1011 13 vg / kg / mouse. Mice were sacrificed 4 weeks post-injection, and multiple organs were collected for evaluation of transduction by native fluorescence or immunohistochemistry (IHC). Figures 2A-2B Representative images showing mCherry expression of AAV9 ( Figure 2A ) and AAV.cc47 ( Figure 2B ) in heart vibratome sections 24 hours after fixation with 4% PFA are provided. Figure 2CWe provide a quantitative analysis of corrected total fluorescence, in which mice infected with AAV.cc47 exhibit more robust mCherry expression in cardiac tissue compared to mice infected with the AVV9 vector. Figure 2D The vector biodistribution of AAV9 and AAV.cc47 in mouse heart tissue is provided. Figures 6A-6C Provided display of AAV9 in cardiac vibratory slices 24 hours after fixation with 4% PFA ( Figure 6A ), AAV.cc81 ( Figure 6B ) and AAV.cc84 ( Figure 6C A representative image of GFP expression. Figure 6D Provides a quantitative analysis of corrected total fluorescence, in which mice infected with AAV.cc84 exhibited more robust GFP expression in cardiac tissue compared to mice infected with AAV.cc81 or AVV9 vectors. Figure 26A Representative images showing mCherry expression of AAV9 and AAV.cc44 in cardiac vibratory slices fixed with 4% PFA 24 hours later are provided. Figure 26B Provides quantitative analysis of corrected total fluorescence.

[0263] Figures 3A-3B Provided a display of AAV9 ( ) in vibratory microtome sections of skeletal muscle 24 hours after fixation with 4% PFA. Figure 3A ) and AAV.cc47 ( Figure 3B A representative image of mCherry expression. Figure 3C A quantitative analysis of corrected total fluorescence was provided, in which mice infected with AAV.cc47 showed more robust mCherry expression in skeletal muscle compared to mice infected with the AVV9 vector. Figures 7A-7B Provides a display of AAV9 ( ) in vibratory microtome sections of skeletal muscle 24 hours after fixation with 4% PFA. Figure 7A ) and AAV.cc81 ( Figure 7B A representative image of GFP expression. Figure 7C Provides a quantitative analysis of corrected total fluorescence, in which mice infected with AAV.cc81 exhibit more robust GFP expression in skeletal muscle compared to mice infected with the AVV9 vector. Figure 26C Representative images of mCherry expression of AAV9 and AAV.cc44 in vibratory microtome sections of skeletal muscle 24 hours after fixation with 4% PFA are provided. Figure 26D Provides quantitative analysis of corrected total fluorescence.

[0264] Figures 4A-4B Provided images showing AAV9 ( ) in vibratory slices of liver 24 hours after fixation with 4% PFA. Figure 4A ) and AAV.cc47 (Figure 4B Representative images of mCherry expression in liver tissue of mice infected with AAV9 and AAV.cc47. Figure 4C Quantitative analysis of corrected total fluorescence is provided. Figure 4D Vector biodistribution of AAV9 and AAV.cc47 in mouse liver tissue is provided. Figures 8A-8C Representative images showing GFP expression in vibratome sections of liver 24 hours after fixation with 4% PFA are provided for AAV9 Figure 8A ), AAV.cc81 Figure 8B ), and AAV.cc84 Figure 8C . Figure 8D Quantitative analysis of corrected total fluorescence is provided, wherein mice infected with AAV.cc84, AAV.cc81, or AVV9 vectors do not show robust GFP expression in the liver. Figure 27A Representative images showing mCherry expression in vibratome sections of liver 24 hours after fixation with 4% PFA are provided for AAV9 and AAV.cc44, and Figure 27B Quantitative analysis of corrected total fluorescence is provided.

[0265] Figures 5A-5B Representative images showing mCherry expression in vibratome sections of kidney tissue 24 hours after fixation with 4% PFA are provided for AAV9 Figure 5A ) and AAV.cc47 Figure 5B . Figure 5C Quantitative analysis of corrected total fluorescence is provided, wherein mice infected with AAV.cc47 have more robust mCherry expression in the kidney compared to mice infected with AVV9 vectors. Figures 9A-9B Representative images showing GFP expression in vibratome sections of skeletal muscle 24 hours after fixation with 4% PFA are provided for AAV9 Figure 9A ) and AAV.cc81 Figure 9B . Figure 9C Quantitative analysis of corrected total fluorescence is provided, wherein mice infected with AAV.cc81 have more robust GFP expression in the skeletal muscle compared to mice infected with AVV9 vectors. Figure 27C Representative images showing mCherry expression in vibratome sections of kidney tissue 24 hours after fixation with 4% PFA are provided for AAV9 and AAV.cc47, and Figure 27D Quantitative analysis of corrected total fluorescence is provided.

[0266] At 5 x 10 13Brain sections were harvested from mice 4 weeks post-intravenous injection of a dose of vg / kg / mouse of either self-complementary AAV9 or one of the ccAAV vectors and examined for AAV vector expression in specific sections of the brain using immunohistochemistry to detect mCherry or GFP. As shown in FIGS. 1-4, all vectors showed localization in brain tissue, however, the robustness of ccAAV vector expression varied by brain region depending on the variant type. Figures 10A-10E and Figures 28A-28C As shown, all vectors showed localization in brain tissue, however, the robustness of ccAAV vector expression varied by brain region depending on the variant type.

[0267] In summary, the data in this example show that the evolved capsid variant proteins enriched in CNS tissue have improved tropism for the brain even after systemic injection into mice. In addition, the evolved ccAAV vectors show strong expression in other non-CNS tissues including heart, skeletal muscle and to some extent the liver. The surprising finding is that the evolved AAV.cc47 vector with only an amino acid substitution at VR4 shows high mCherry transduction expression levels in the kidney. To date, there is no known AAV vector that is capable of high transduction efficiency in the kidney and of the ccAAV vectors tested, only AAV.cc47 shows this phenotype (AAV.cc81 and AAVcc.84 - both with amino acid substitutions within VR8 - are not able to transduce express in the kidney).

[0268] Example 3. In vivo characterization of recombinant AAV in pigs

[0269] The ccAAV vectors packaged with a fluorescent reporter used in Example 2 herein were also used in Example 3. Here, a 3 week old, newly weaned piglet weighing approximately 7 kg was administered by intrathecal infusion with a dose of approximately 3 x 1011vg / 7 kg piglet (in 2 ml) of either self-complementary AAV9 or ccAAV vectors. Four weeks post-injection, the piglet was sacrificed and brain, spinal cord, heart and liver were harvested. Transduction was evaluated by native fluorescence or by IHC performed as described herein. 13 Example 3.

[0270] Prior to subjecting the tissue to IHC, pig brain sections were dissected and stored in 4% PFA in order to evaluate the transduction efficiency of AAV.cc47 ( Figures 11A-11G ) and AAV.cc84 ( Figures 12A-12G ) in frontal cortex, parietal cortex, thalamus, occipital cortex, brainstem, cerebellum and midbrain. Pig spinal cord sections were also harvested and subjected to IHC to evaluate the transduction efficiency of AAV.cc47 ( Figure 13A ) and AAV.cc84 ( Figure 13B ) in the tissue. To examine more closely, the white matter and the gray matter of the pig spinal cord were also examined for AAV.cc47 ( Figure 13C and 13E ) and AAV.cc84 ( Figure 13D and 13FTransduction efficiency was assessed by observing mCherry or GFP fluorescence at magnification.

[0271] The pig's heart and liver tissue were also harvested at the time of sacrifice and subjected to IHC to evaluate AVV.cc47. Figures 14A-14C ) and AVV.cc84 ( Figures 14D-14F The transduction efficiency of ).

[0272] Example 4. In vivo characterization of recombinant AAV in non-human primates (NHPs)

[0273] The ccAAV vector used for packaging fluorescent reporter genes in Examples 2 and 3 of this paper was also used in Example 4. In this paper, a two-year-old rhesus monkey (NHP) weighing approximately 3 kg was packaged using a 3.5 x 10⁻⁶ vector. 12 Intracision injection of complementary AAV9, AAV.cc47, or AAV.cc84 vectors at a dose of vg / kg into the cerebral cisterns. Two weeks post-injection, NHPs were bagged, and the brain, liver, heart, and spinal cord were harvested. Regarding the liver (… Figure 15A ) and heart ( Figure 15C AAV9 in the liver () Figure 15B ) and heart ( Figure 15D IHC analysis of mCherry was performed using AAV.cc47 in the file. Figure 15E The vector biodistribution of AAV9 and AAV.cc47 vectors in the liver and heart is shown.

[0274] Next, IHC analysis of mCherry for AAV9 and AAV.cc47 and IHC analysis of GFP for AAV.cc84 were performed in NHP brains. Compared with sham-treated brain slices ( Figure 16A AAV9, AAV.cc47, and AAV.cc84 all showed some degree of transduction in brain tissue. Figures 16B-16D The data indicate that: (1) the cross-species capsid has a unique biological distribution pattern and is different from AAV9; (2) AAV.cc47 appears to diffuse more deeply into brain tissue and transduce more cells; and (3) AAV.cc84 also diffuses well into tissues but transduces fewer (more specific) cells.

[0275] Example. 5AAVcc47 heart transduction

[0276] To validate AAVcc47 cardiac transduction, human iPSC cardiomyocytes were transduced with AAV9 or AAVcc47 packaged with GFP driven by the Cbh promoter. Figure 17A Next, the area percentage of GFP+ cells in multiple images was quantified ( Figure 17B In a human heart patch mouse model, AAV9 or AAVcc47 packaged with CBh:GFP was injected intravenously.Figure 17C ), and fluorescent imaging of the cardiac patch was performed ( Figure 17D ). AAV9 and AAV.cc47 were again administered to the human cardiac patch mouse model via i.v., this time delivering GFP under the control of an injury-inducible promoter following myocardial infarction. Immunofluorescence for troponin T (red) and GFP (green) was performed on heart tissue harvested from mice post-injection ( Figure 17E ).

[0277] Example 6. Recombination of cre with ccAAV vectors

[0278] Ai9 male and female mice were injected i.v. with single-stranded AAV9 or ccAAV vectors at a dose of 1 x 1010 12 vg / kg (N=3). Animals were sacrificed 4 weeks post-injection, multiple organs were collected, and transduction was evaluated by native fluorescence or IF. Figures 18A-18D Representative images showing native tdTomato fluorescence following i.v. administration of AAV9 or ccAAV vectors in the mouse heart, Figure 18E showing biodistribution of AAV9, AAV.cc47, and AAV.cc84 vectors in heart tissue. Figures 19A-19D Representative images showing native tdTomato fluorescence following i.v. administration of AAV9 or ccAAV vectors in the mouse liver, and Figure 19E showing biodistribution of AAV9, AAV.cc47, and AAV.cc84 vectors in liver tissue. Figures 20A-20D Representative images showing native tdTomato fluorescence following i.v. administration of AAV9 or ccAAV vectors in the mouse lung, co-stained with DAPI (nuclear marker) and SPC, and Figure 20E showing biodistribution of AAV9, AAV.cc47, and AAV.cc84 vectors in lung tissue.

[0279] Example 7. CRISPR / Cas9 gene editing with ccAAV vectors

[0280] A dual vector strategy was employed using a first vector and a second vector, where the first vector had a truncated CB promoter driving SaCas9 and a U6 promoter driving one sgRNA, and the second vector had the same design as the second sgRNA ( Figure 21A ). Male and female Ai9 mice were injected i.v. with a dual single-stranded vector consisting of sgRNA1 and sgRNA2 mixed 50:50 with AAV9 or ccAAV at a dose of 2 x 1010 12 vg (N=6). Animals were sacrificed 4 weeks post-injection, multiple organs were collected, and transduction was evaluated by native fluorescence or immunofluorescence (IF).

[0281] Natural tdTomato fluorescence was evaluated in the liver and heart of Ai9 mice after administration of AAV9 or AAV.cc47. Figure 21B Gene editing efficiency was determined by calculating the total number of tdTomato+ cells and dividing it by the total number of DAPI+ cells. Figure 21C PCR editing assays were performed on liver and heart tissues. Figure 21D ).

[0282] Validated using the same dual-vector strategy with a CB promoter that drives SaCas9 expression. Figures 21A-21D The results showed that each vector had a guide targeting the Rosa26 locus. These vectors were mixed in equal volumes and then... 14 Ai9 mice were intravenously injected with a dose of vg / kg. Ai9 liver sections were sliced ​​and imaged for expression of natural tdtomato (vg / kg). Figure 22A Gene editing efficiency was quantified by counting the total number of tdtomato+ cells and normalizing it to the total number of Dapi+ cells. Figure 22B Ai9 heart slices were sliced ​​and imaged for expression of natural tdtomato (). Figure 22C ).

[0283] As a means of quantifying CRISPR / Cas9CB, fluorescence intensity was measured from multiple images to quantify native tdtomato expression in the heart and liver tissues of male and female Ai9 mice. Figures 23A-23F In addition, in Figure 24A and 24B The relative PCR band intensity was measured (unedited simulated samples vs. edited experimental samples). Furthermore, in... Figure 25A and 25B In this study, editing efficiency is quantified using the following formula: Editing efficiency (%) = #Red blood cells (count w / image j (liver) or manual (heart)) / #DAPI-stained cell nuclei.

[0284] Example 8. Administration of ccAAV via intraventricular (ICV) injection

[0285] Using self-complementary AAV9 or ccAAV carriers at 1x10 10 p0C57 / BL6 neonatal mice were injected intraventricularly (ICV) at a dose of vg (N=4), and the constructs of the self-complementary AAV9 or ccAAV vector were described in Figure 29A and 29B Animals were euthanized 4 weeks after injection.

[0286] Gene expression was reported using natural fluorescence detection. Figures 30A-30F The image shows ICV injection of AAV9mCherry (Figure 30A ), AAV.cc44 ( Figure 30B ), AAV.cc47 ( Figure 30C ), AAV9eGFP ( Figure 30D ), AAV.cc81 ( Figure 30E ), or AAV.cc84 ( Figure 30F ) in mouse brains.

[0287] Brain tissues harvested from mice 4 weeks post-infection were also subjected to immunofluorescence (IF). The tissues were stained with DAPI (4', 6-diamidino-2-phenylindole) to visualize nuclear DNA; anti-NeurN antibody (a-NeurN) to visualize neuronal nuclei specifically; and either anti-mCherry (a-mCherry) or anti-eGFP (a-GFP) antibodies to visualize the reporter gene expression of the injected AAV vectors. Images of the resulting IF for each antibody were collected and merged to detect co-localization. Figures 31A-31B and Figures 32A-32B Representative images of cerebellum, hippocampus, and cerebral cortex regions of brains of mice harvested 4 weeks post-injection of AAV9eGFP ( Figure 31A ), AAV.cc84 ( Figure 31B ), AAV9mCherry ( Figure 32A ), or AAV.cc47 ( Figure 32B ) are shown. Quantification of the number of eGFP and NeurN positive stained neurons in the cerebellum ( Figure 31C ), hippocampus ( Figure 31D ), and cerebral cortex ( Figure 31E ) of mice injected with AAV9 (eGFP) or AAV.cc84. Quantification of the number of mCherry and NeurN positive stained neurons in the cerebellum ( Figure 32C ), hippocampus ( Figure 32D ), and cerebral cortex ( Figure 32E ) of mice injected with AAV9 (mCherry) or AAV.cc47.

[0288] Those skilled in the art will readily understand that the disclosure is well adapted to attain the ends and objectives hereinabove set forth, as well as those inherent to it, and that it is not limited to those details described hereinabove because various modifications can be made and still be within the spirit of the disclosure, as will be apparent to those skilled in the art. The disclosure described herein is to be considered merely as illustrative of the presently preferred embodiments and is not intended to limit the scope of the disclosure to the precise embodiments described. Changes and modifications can be suggested to one skilled in the art, and it is intended to encompass such changes and modifications as fall within the scope of the appended claims.

[0289] No admission is made that any reference in this specification to any non-patent or patent document constitutes prior art. In particular, this statement is not to be construed as an admission that any of the documents discussed in this specification is a prior art document with respect to any patentable matter disclosed herein. Any discussion of the references states what their authors assert and the applicant reserves the right to challenge the accuracy and pertinency of any of the documents discussed in this specification; and to otherwise raise outstanding questions regarding the documents discussed in this specification and / or the documents themselves. All references cited herein are fully incorporated by reference in their entirety.

[0290] In the event of any discrepancy between any definitions and / or descriptions found in cited references, the present disclosure shall prevail.

[0291] Numbered embodiments

[0292] While the appended claims are presented with specificity and detail, the following numbered embodiments are also encompassed by this text and form part of the disclosure:

[0293] 1. A recombinant AAV vector comprising 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.

[0294] 2. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28.

[0295] 3. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0296] 4. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28; and wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0297] 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 that is at least 90% or at least 95% identical thereto.

[0298] 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 that has 1-10, 11-20, 20-30, or 30-50 amino acid substitutions relative thereto.

[0299] 7. The recombinant AAV vector of any one of embodiments 1-6, wherein the AAV vector comprises a vector genome.

[0300] 8. The recombinant AAV vector of embodiment 7, wherein the vector genome is encapsidated by an AAV capsid comprising an AAV capsid protein variant.

[0301] 9. The recombinant AAV vector of embodiments 7 or 8, wherein the vector genome comprises a first inverted terminal repeat sequence (ITR) and a second ITR.

[0302] 10. The recombinant AAV vector of embodiment 9, wherein the vector genome comprises a transgene located between the first ITR and the second ITR.

[0303] 11. The recombinant AAV vector of embodiment 10, wherein the transgene encodes a therapeutic RNA.

[0304] 12. The recombinant AAV vector of embodiment 10, wherein the transgene encodes a therapeutic protein.

[0305] 13. The recombinant AAV vector of embodiment 10, wherein the transgene encodes a gene editing molecule.

[0306] 14. The recombinant AAV vector of embodiment 13, wherein the gene editing molecule is a nuclease.

[0307] 15. The recombinant AAV vector of embodiment 14, wherein the nuclease is a Cas9 nuclease.

[0308] 16. The recombinant AAV vector of embodiment 14, wherein the nuclease is a Casl2a nuclease.

[0309] 17. The recombinant AAV vector of embodiment 13, wherein the gene editing molecule is a single guide RNA (sgRNA).

[0310] 18. An AAV capsid protein variant comprising a peptide having the sequence of any one of SEQ ID NOs: 2-19.

[0311] 19. An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28.

[0312] 20. An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0313] 21. An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein the amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 20-28; and wherein the amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are substituted with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0314] 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.

[0315] 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 relative thereto.

[0316] 24. An AAV capsid comprising the AAV capsid protein variant of any one of embodiments 18-23.

[0317] 25. The AAV capsid of embodiment 24, wherein the AAV capsid comprises about 60 copies of the AAV capsid protein variant or fragment thereof.

[0318] 26. The AAV capsid of embodiment 25, wherein the AAV capsid protein variant is arranged in T = 1 icosahedral symmetry.

[0319] 27. A recombinant AAV vector comprising the AAV capsid variant of any one of embodiments 18-23 or the AAV capsid of any one of embodiments 24-26.

[0320] 28. A pharmaceutical composition comprising the recombinant AAV vector of any one of embodiments 1-17 and 27 and at least one pharmaceutically acceptable carrier.

[0321] 29. A method of introducing a recombinant AAV vector into a target cell, the method comprising contacting the target cell with the recombinant AAV vector of any one of embodiments 1-17 and 27 or the pharmaceutical composition of embodiment 28.

[0322] 30. A method of delivering a transgene to a target cell in a subject, the method comprising administering to the subject the recombinant AAV vector of any one of embodiments 1-17 and 27 or the pharmaceutical composition of embodiment 28.

[0323] 31. The method of any one of embodiments 29 and 30, wherein the target cell is a kidney cell.

[0324] 32. A method of evolving a new adeno-associated virus strain, comprising passaging an AAV library across a plurality of mammalian species.

[0325] 33. The method according to embodiment 32, wherein the AAV library comprises a plurality of recombinant AAV vectors, wherein each recombinant AAV vector comprises a capsid protein variant comprising one or more amino acid mutations relative to a wild-type AAV capsid protein.

[0326] 34. The method of embodiment 33, wherein 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).

[0327] 35. The method of embodiment 34, wherein the one or more amino acid mutations are located in a region corresponding to amino acids 452-458 of SEQ ID NO: 1 or 586-592 of SEQ ID NO: 1, or the mutations are found in both regions corresponding to amino acids 452-458 and 586-592 of SEQ ID NO: 1.

[0328] 36. The method of any one of embodiments 31-35, wherein the method comprises administering a first AAV library to a first mammalian species.

[0329] 37. The method of embodiment 36, wherein the AAV of the first AAV library present in one or more target tissues from the first mammalian species are sequenced and used to generate a second AAV library.

[0330] 38. The method of embodiment 37, wherein the second AAV library is administered to a second mammalian species, wherein the first mammalian species and the second mammalian species are different.

[0331] 39. The method of embodiment 38, wherein AAVs from a second AAV library present in one or more target tissues of a second mammalian species are sequenced.

[0332] 40. The method of 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 lupus familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human).

[0333] 41. The method of embodiment 40, wherein the one or more target tissues of the first mammalian species are selected from the group consisting of spinal cord, dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0334] 42. The method of embodiment 40, wherein the one or more target tissues of the second mammalian species are selected from the group consisting of spinal cord, dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0335] 43. A recombinant adeno-associated virus (AAV) comprising a capsid protein variant evolved using the method of any one of embodiments 31-42.

[0336] 44. The recombinant AAV of embodiment 43, wherein the AAV has improved gene transfer efficiency in one or more mammalian species relative to a recombinant AAV having a capsid protein that is otherwise identical except that it lacks one or more amino acid substitutions.

[0337] 45. The recombinant AAV of embodiment 44, wherein the improved gene transfer efficiency occurs in one or more of Mus musculus (mouse), Sus scrofa (pig), Canis lupus familiaris (dog), non-human primate (Macaca, Macaca), or Homo sapiens (human).

[0338] 46. The recombinant AAV of embodiments 43-45, wherein the improved gene transfer efficiency occurs in one or more cell types or tissues selected from the group consisting of spinal cord, dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver.

[0339] 47. The recombinant AAV of embodiment 46, wherein the improved gene transfer efficiency occurs in kidney cells or kidney tissue.

[0340] 48. 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 embodiments 1-17, 27, and 43-47 or the pharmaceutical composition of embodiment 28.

[0341] 49. The method of embodiment 48, wherein the subject has a kidney disease or kidney disorder.

Claims

1. An AAV vector containing an AAV capsid protein, wherein the capsid protein comprises the sequence of SEQ ID NO:

8.

2. The AAV vector of claim 1, wherein the AAV vector comprises a vector genome, wherein the vector genome is encapsulated by an AAV capsid containing an AAV capsid protein.

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

4. The AAV vector of claim 3, wherein the vector genome contains transgenes located between the first ITR and the second ITR.

5. The AAV vector according to claim 4, wherein the transgene encodes therapeutic RNA.

6. The AAV vector of claim 4, wherein the transgene encodes a therapeutic protein.

7. The AAV vector according to claim 4, wherein the transgene encodes a nuclease.

8. The AAV vector according to claim 7, wherein the nuclease is a Cas9 nuclease.

9. The AAV vector according to claim 7, wherein the nuclease is a Cas12a nuclease.

10. The AAV vector according to claim 7, wherein the transgene encodes a single guide RNA (sgRNA).

11. AAV capsid protein, consisting of the sequence of SEQ ID NO:

8.

12. A pharmaceutical composition comprising the AAV carrier according to claim 4 and at least one pharmaceutically acceptable carrier.

13. Use of the AAV vector of claim 4 in the preparation of a medicament or medicament composition for introducing transgenes into cells.

14. Use of the AAV carrier of claim 4 in the preparation of a medicament or pharmaceutical composition for treating kidney disease.

15. The use according to claim 13, wherein the cell is a kidney cell.

16. The use according to claim 13, wherein the cells are in the spinal cord, dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine or liver tissue.

17. The use according to claim 14, wherein the kidney disease is Allport syndrome, polycystic kidney disease, nephrolithiasis, Fabry disease, or steroid-resistant nephrotic syndrome.

18. The AAV vector of claim 4, further comprising a promoter operatively linked to the transgene.

19. A plasmid comprising an AAV cap gene encoding the AAV cap protein according to claim 11.

20. The plasmid of claim 19, further comprising the AAV rep gene.

21. A cell line that stably expresses the AAV capsid protein according to claim 11.

22. The cell line according to claim 21, further expressing the AAV rep gene.

Images