Novel adeno-associated virus (AAV) vectors, AAV vectors with reduced capsid deamidation, and uses thereof

Abstract

Compositions comprising a mixed population of recombinant adeno-associated virus (rAAV) and methods of reducing deamidation in the AAV capsid of rAAV are provided.SOLUTION: Provided is a composition comprising a mixed population of recombinant adeno-associated viruses (rAAVs), wherein each of the rAAVs comprises (a) an AAV capsid comprising about 60 capsid vp1, vp2, and vp3 proteins, and (b) a vector genome in the AAV capsid, wherein the vector genome comprises AAV inverted terminal repeat sequences and a non-AAV nucleotide sequence encoding a product operably linked to sequences directing expression of the product in host cells.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] Description of Federally Sponsored Research This invention was made with government support under Grant No. P01HL059407 awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0002] The adeno-associated virus (AAV) capsid is an icosahedral structure consisting of 60 viral protein (VP) monomers (VP1, VP2, and VP3) in a 1:1:10 ratio (Xie Q, et al. Proc Natl Acad Sci USA. 2002;99(16):10405-10). The entire VP3 protein sequence (519 aa) is contained within the C-terminus of both VP1 and VP2, and the shared VP3 sequence is primarily responsible for the overall capsid structure. Due to the structural flexibility of the VP1 / VP2 unique region and the low representation of VP1 and VP2 monomers compared to VP3 monomers in assembled capsids, VP3 is the only capsid protein to be resolved via X-ray crystallography (Nam HJ, et al. J Virol. 2007;81(22):12260-71). VP3 contains nine hypervariable regions (HVRs), which are the main source of sequence diversity among AAV serotypes (Govindasamy L, et al. J Virol. 2013;87(20):11187-99). Given their flexibility and location on the capsid surface, HVRs are largely involved in interactions with target cells and the immune system (Huang LY, et al. J Virol. 2016;90(11):5219-30, Raupp C, et al. J Virol. 2012;86(17):9396-408). Although structures of several serotypes have been published (Structural entries for AAV2, AAVrh.8, AAV6, AAV9, AAV3B, AAV8, and AAV4 from the Research Collaboratory for Structural Bioinformatics (RCSB) database, Protein Data Bank (PDB) numbers 1LP3, 4RSO, 4V86, 3UX1, 3KIC, 2QA0, and 2G8G, respectively), there is very little information in the literature about surface modifications of these capsids. Studies suggest that intracellular phosphorylation of capsids occurs at specific tyrosine residues (Zhong L, et al. Virology. 2008;381(2):194-202). Despite predicted glycosylation sites in the major VP3 sequence, glycosylation events in AAV2 have not been identified (Murray S, et al. J Virol. 2006;80(12):6171-6, Jin X, et al. Hum Gene Ther Methods. 2017;28(5):255-267), and other AAV serotypes have not yet been evaluated for capsid glycosylation.

[0003] AAV gene therapy vectors have not received the molecular scrutiny typically associated with the development and production of recombinant protein therapeutics. AAV capsid post-translational modifications (PTMs) have remained largely unexplored, and little is known about their potential impact on function or strategies for controlling PTM levels in manufactured AAV therapeutics.

[0004] The diversity of post-translational modifications of non-gene therapy protein therapeutics complicates their development as drugs. Jenkins, N, Murphy, L, and Tyther, R (2008) Post-translational modifications of recombinant proteins:significance for b Biopharmaceuticals. Mol Biotechnol 39:113-118; Houde, D, Peng, Y, Berkowitz, SA, and Engen, JR (2010). Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol Cell Proteomics 9:1716-1728. For example, deamidation of selected amino acids modulates the stability and immune response of recombinant protective antigen-based anthrax vaccines. (Powell BS, et al. Proteins. 2007;68(2):45879; Verma A, et al. Clin Vaccine Immunol. 2016;23(5):396-402). In some cases, this process is catalyzed by viral or bacterial deamidases and modulates host cell signaling pathways or innate immune responses (Zhao J, et al. J Virol. 2016;90(9):4262-8; Zhao J, et al. Cell Host Microbe. 2016;20(6):770-84). More generally, endogenous deamidation is an enzyme-independent spontaneous process. Although the purpose of spontaneous deamidation is not fully understood, previous studies have suggested that this event serves as a molecular clock to indicate the relative age of proteins and regulate their metabolic turnover (Robinson NE and Robinson AB. Proc Natl Acad Sci USA. 2001;98(3):944-9).

[0005] Deamidation occurs when the amide group of asparagine, or less frequently glutamine, undergoes nucleophilic attack from the adjacent nitrogen atom, resulting in the loss of the amide group. This process results in a succinimidyl intermediate (Yang H and Zubarev RA. Electrophoresis. 2010;31(11):1764-72), which decomposes via hydrolysis into a mixture of aspartic acid and isoaspartic acid (or glutamic acid and isoglutamic acid) (Catak S, et al. J Phys Chem A. 2009;113(6):1111-20). Studies of short synthetic peptides suggest that this hydrolysis results in a 3:1 mixture of isoaspartic acid to aspartic acid (Geiger T. and Clarke SJ Biol Chem. 1987;262(2):785-94).

[0006] There continues to be a need for compositions comprising AAV-based constructs for delivering heterologous molecules that have stable receptor binding and / or stable capsids, avoid neutralizing antibodies, and / or retain purity upon storage. Summary of the Invention

[0007] In one embodiment, a composition comprises a mixed population of recombinant adeno-associated viruses (rAAVs), each of the rAAVs comprising (a) about 60 capsid vp1 proteins, vp2 proteins, and vp3 proteins, wherein the vp1, vp2, and vp3 proteins are selected from the group consisting of a heterogeneous population of vp1 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 proteins produced from a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence, a heterogeneous population of vp3 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 proteins produced from a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence, a heterogeneous population of vp1 proteins produced from a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence, a heterogeneous population of vp2 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 ...2 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 A composition is provided that includes: (a) a heterogeneous population of vp3 proteins produced from a nucleic acid sequence encoding a vp3 amino acid sequence, wherein the vp1, vp2, and vp3 proteins include a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in asparagine-glycine pairs in the AAV capsid, and optionally further subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (b) a vector genome in the AAV capsid, wherein the vector genome comprises a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell. The resulting rAAVs are derived from a production system that uses a single AAV capsid nucleic acid sequence encoding the predicted AAV VP1 amino acid sequence of one AAV type. However, the manufacturing and production process provides a heterogeneous population of the above capsid proteins. In certain embodiments, the composition is as described in this paragraph, except that the rAAV is not AAVhu68. In certain embodiments, the composition is as described in this paragraph, except that the rAAV is not AAV2.

[0008] In certain embodiments, the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid / isoaspartic acid pair, or a combination thereof. In certain embodiments, the capsid further comprises a deamidated glutamine that is deamidated to (α)-glutamic acid, γ-glutamic acid, an interconverted (α)-glutamic acid / γ-glutamic acid pair, or a combination thereof.

[0009] In certain embodiments, methods are provided for reducing deamidation of AAV capsids, comprising producing AAV capsids from nucleic acid sequences comprising modified AAV vp codons, the nucleic acid sequences comprising glycine codons independently modified at one to three of asparagine-glycine pairs relative to a reference AAV vp1 sequence, such that the modified codons encode amino acids other than glycine.

[0010] In another embodiment, a method for reducing deamidation of an AAV capsid is provided, comprising producing an AAV capsid from a nucleic acid sequence comprising modified AAV vp codons, the nucleic acid sequence comprising asparagine codons independently modified with at least one asparagine-glycine pair relative to a reference AAV vp1 sequence, such that the modified codon encodes an amino acid other than asparagine.

[0011] Methods for increasing AAV titer, efficacy, and / or transduction are provided. The methods include producing AAV capsids from a nucleic acid sequence containing at least one AAV vp codon modified to change the asparagine or glycine of at least one asparagine-glycine pair in the capsid to a different amino acid. In certain embodiments, the modified codon is within the v2 and / or vp3 region. In certain embodiments, the asparagine-glycine pair in the vp1 unique region is retained in the modified rAAV. In certain embodiments, nucleic acid molecule sequences encoding these mutant AAV capsids are provided.

[0012] In certain embodiments, the deamidation site (e.g., an asparagine-glycine pair or Gln) is modified at a position other than (a) N57, N263, N385, N514, and / or N540 of SEQ ID NO:6 (encoding AAV8 vp1) based on the numbering of AAV8 vp1 with the first M for an AAV8 capsid, (b) N57, N329, N452, and / or N512 based on the numbering of SEQ ID NO:7 (encoding AAV9 vp1) with the first M for an AAV9 capsid, or (c) N263, N385, and / or N514 based on the numbering of SEQ ID NO:112 (encoding AAVrhlO vp1) with the first M for an AAVrhlO capsid. In certain embodiments, the modified deamidation site is selected from the Table F or Table G sites. In certain embodiments, the modified deamidation site is selected from the sites of Table F or Table G, excluding positions (a) through (c) above. In certain embodiments, the deamidation site (e.g., an asparagine-glycine pair or Gln(Q)) is selected from (a) for AAV1 capsids, N57, N383, N512, and / or N718, based on the numbering of SEQ ID NO: 1, based on the numbering of the predicted vp1 amino acid sequence with the first M; (b) for AAV3B capsids, N57, N382, N512, and / or N718, based on the numbering of SEQ ID NO: 2, based on the numbering of the predicted vp1 amino acid sequence with the first M; or N718; (c) for AAV5 capsids, N56, N347, N347, and / or N509, based on the numbering of the predicted vp1 amino acid sequence with the first M and with reference to the numbering of SEQ ID NO: 3; (d) for AAV7 capsids, N41, N57, N384, and / or N514, based on the numbering of the predicted vp1 amino acid sequence with the first M and with reference to the numbering of SEQ ID NO: 4; (e) for AAVrh32.33 capsids, N57, N264, N292, and / or N318, based on the numbering of the predicted vp1 amino acid sequence with the first M and with reference to the numbering of SEQ ID NO: 5; or (f) for AAV4 capsids, modified at a position other than N56, N264, N318, and / or N546, based on the numbering of the predicted vp1 amino acid sequence with the first M and with reference to the numbering of SEQ ID NO: 111. In certain embodiments, the modified deamidation site is selected from a site in Table A, Table B, Table C, Table D, Table E, Table F, or Table G. In certain embodiments, the modified deamidation site excludes positions (a) through (f) above.

[0013] In certain embodiments, the method comprises selecting a sequence from another AAV based on the numbering of AAV8 or based on an alignment of the selected sequence with AAV8, such as AAV8 G264A / G515A (SEQ ID NO:21), AAV8G264A / G541A (SEQ ID NO:23), AAV8G515A / G541A (SEQ ID NO:25), or AAV8 G264A / G515A / G541A (SEQ ID NO:27), AAV8 G264A / G541A / N499Q (SEQ ID NO:115), (c) AAV8 G264A / G541A / N459Q (SEQ ID NO:116), (d) AAV8 G264A / G541A / N305Q / N459Q (SEQ ID NO:117), (e) AAV8 G264A / G541A / N305Q / N499Q (SEQ ID NO: 118), AAV8 and generating a recombinant AAV having a selected mutant AAV8 capsid with the mutations G264A / G541A / N459Q / N499Q (SEQ ID NO: 119), or AAV8 G264A / G541A / N305Q / N459Q / N499Q (SEQ ID NO: 120). In certain embodiments, the method includes generating a rAAV having a mutant AAV9 capsid selected from AAV9 G330 / G453A (SEQ ID NO: 29), AAV9 G330A / G513A (SEQ ID NO: 31), AAV9 G453A / G513A (SEQ ID NO: 33), and / or AAV9 G330 / G453A / G513A (SEQ ID NO: 35).

[0014] In certain embodiments, nucleic acid molecule sequences encoding these mutant AAV capsids are provided. In certain embodiments, the nucleic acid sequences are provided, for example, in SEQ ID NO:20 (AAV8 G264A / G515A), SEQ ID NO:22 (AAV8 G264A / G541A), SEQ ID NO:24 (AAV8 G515A / G541A), or SEQ ID NO:26 (AAV8 G264A / G515A / G541A). In certain embodiments, the nucleic acid sequences are provided, for example, in SEQ ID NO:28 (9G330AG453A), SEQ ID NO:30 (9G330AG513A), SEQ ID NO:32 (9G453AG513A), or SEQ ID NO:34 (9G330AG453AG513A). In certain embodiments, other AAVs can be mutated to have these or corresponding NG pair changes based on alignment with AAV 9.

[0015] Compositions are provided that include a population of rAAVs with increased titer, efficacy, or transduction. In certain embodiments, the compositions include rAAVs having capsids modified to have reduced total deamidation compared to rAAVs having a capsid deamidation pattern according to any one of Table A (AAV1), Table B (AAV3B), Table C (AAV5), Table D (AAV7), Table E (AAVrh32.33), Table F (AAV8), Table G (AAV9), or Table H (AAVhu37). In certain embodiments, the rAAVs are unmodified at the highly deamidated positions identified herein.

[0016] These and other aspects of the present invention will become apparent from the following detailed description of the invention. Deaf. [Brief explanation of the drawings]

[0017] [Figure 1-1] Figures 1A-1G show electrophoretic analysis of AAV8 VP isoforms. (Figure 1A) The diagram illustrates the mechanism by which an asparagine residue undergoes nucleophilic attack by an adjacent nitrogen atom, forming a succinimidyl intermediate. This intermediate then undergoes hydrolysis, decomposing into a mixture of aspartic acid and isoaspartic acid. The beta carbon is thus labeled. The diagram was generated in BIOVIA Draw 2018. (Figure 1B) 1 μg of AAV8 vector was run on a denaturing one-dimensional SDS-PAGE. (Figure 1C) The isoelectric points of the carbonic anhydrase pI marker spots are shown. (Figure 1D) 5 μg of AAV8 vector was analyzed by two-dimensional gel electrophoresis and stained with Coomassie Blue. Spots 1-20 are carbamylated carbonic anhydrase pI markers. Boxed regions are a = VP1, b = VP2, c = VP3, and d = internal tropomyosin marker (arrow: tropomyosin spot with MW = 33 kDa and pI = 5.2). Isoelectric focusing was performed in the pI range of 4-8 (Figure 1E-1G). Results of isoelectric focusing performed in the pI range of 4-8. 1e11 GC of wtAAV8 (Figure 1E) or mutant (Figure 1F and Figure 1G) vectors were analyzed by 2D gel electrophoresis and stained with Sypro Ruby. Protein labels: A = VP1, B = VP2, C = VP3, D = chicken egg white conalbumin marker, E = turbonuclease marker. Isoelectric focusing was performed in the pI range of 6-10. The major VP1 / 2 / 3 isoform spots are circled, and the migration distance of the major spots of the markers is indicated by vertical lines (turbonuclease = dashed, conalbumin = solid). [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 2-1]Figures 2A-2E show analysis of asparagine and glutamine deamidation in the AAV8 capsid protein. (Figures 2A-2B) Electrospray ionization (ESI) mass spectrometry (ESI) analysis of the 3+ peptide (93-103) containing Asn-94 (Figure 2A) and Asp-94 (Figure 2B) is shown, along with the theoretical and observed masses. (Figures 2C-2D) ESI mass spectrometry analysis of the 3+ peptide (247-259) containing Asn-254 (Figure 2C) and Asp-254 (Figure 2D) is shown, along with the theoretical and observed masses. The observed mass shifts at Asn-94 and Asn-254 were 0.982 Da and 0.986 Da, respectively, compared to a theoretical mass shift of 0.984 Da. (Figure 2E) Deamidation rates at specific asparagine and glutamine residues of interest are shown for AAV8 tryptic peptides purified by different methods. Bars indicating deamidation at asparagine residues with N+1 glycine are shaded. Residues determined to be at least 2% deamidated in at least one preparation analyzed were included. Data are presented as mean ± standard deviation. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 3-1]Figures 3A-E show structural modeling of the AAV8 VP3 monomer and analysis of the deamidation site. (Figure 3A) The AAV8 VP3 monomer (PDB identifier: 3RA8) is shown in a coil representation. Ribbon colors indicate relative flexibility (blue = most rigid / normal temperature factor, red = most flexible / high temperature factor). Spheres indicate residues of interest. The enlarged view shows a ball-and-stick representation of the residue of interest and its surrounding residues, showing the local protein structure (blue = nitrogen, red = oxygen). The underlined residues are from the NG motif. Figures 3B-E show an isoasparagine-based model of a deamidated asparagine with an N+1 glycine. A 2FoFc electron density map (1 sigma level) was generated from the refinement of the AAV8 crystal structure (PDB number: 3RA8) with an asparagine model for (Figure 3B) N410 compared to an isoaspartic acid model for (Figure 3C) N263, (Figure 3D) N514, and (Figure 3E) N540. The electron density map is shown in magenta grid. Beta carbons are labeled as such. Arrows indicate electron density corresponding to the R group of the residue of interest. [Figure 3-2] Same as above. [Figure 4A] Figures 4A-4D show the determination of factors affecting AAV8 capsid deamidation. AAV8 preparations were (Figure 4A) incubated at 70°C for 3 or 7 days, (Figure 4B) exposed to pH 2 or pH 10 for 7 days, or (Figure 4C) prepared for mass spectrometry analysis using DO instead of HO to identify possible sources of deamidation not inherent to AAV capsid formation. (Figure 4D) Dot blot of vectors processed as in Figure 4A using the B1 antibody (reacting to denatured capsids) and an AAV8 structural-specific antibody (reacting to intact capsids) to assess capsid structural integrity. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 5A]Figures 5A-5B show the deamidation frequency of non-AAV proteins. Deamidation rates are shown for two non-AAV recombinant proteins containing NG motifs that are likely to be deamidated: human carbonic anhydrase (Figure 5A) and rat phenylalanine hydroxylase (Figure 5B), for comparison with AAV deamidation rates. [Figure 5B] Same as above. [Figure 6] Figure 6 shows a comparison of AAV8 deamidation rates calculated using data analysis pipelines from two institutions. Deamidation rates at specific asparagine and glutamine residues of interest are shown for AAV8 tryptic peptides evaluated at two different institutions. [Figure 7A] Figures 7A-7C illustrate functional asparagine substitutions at non-NG sites, which exhibit high lot-to-lot variability. (Figure 7A) Titers of wtAAV8 and mutant vectors produced by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). Titers are reported relative to the wtAAV8 control. Transduction efficiency was measured as described in Figure 8B. Titers and transduction efficiency are normalized to the wtAAV8 control value. (Figure 7B) Representative luciferase images at day 14 post-injection are shown for mice receiving wtAAV8.CB7.ffluc and N499Q capsid mutant vectors. (Figure 7C) Luciferase expression at day 14 of the study period from C57BL / 6 mice intravenously injected with wtAAV8 or mutant vectors (n = 3 or 4), measured by luciferase imaging and reported in total flux units. All data are expressed as mean + standard deviation. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8A]Figures 8A and 8B show the results of an in vitro analysis of the impact of genetic deamidation on vector performance. (Figure 8A) Titers of wtAAV8 and genetic deamidation mutant vectors produced by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). Titers are reported relative to the wtAAV8 control. Highly deamidated NG sites (patterned bars), lowly deamidated sites (white bars), and highly variable sites (black bars) are displayed in wtAAV8 and negative controls. (Figure 8B) Transduction efficiencies of firefly luciferase-producing mutant AAV8 vectors reported relative to the wtAAV8 control. Transduction efficiency is measured in luminescence units produced per GC added to HUH7 cells and determined by transducing crude vector at multiple dilutions. Transduction efficiency data are normalized to the wild-type (wt) reference. All data are expressed as mean ± standard deviation. [Figure 8B] Same as above. [Figure 9A] Figures 9A-9D illustrate that vector activity loss over time correlates with progressive deamidation. (Figure 9A) Vector production (DNAseI-resistant genome copies, GC) over a time course of triple-transfected HEK293 cells producing AAV8 vectors packaging a luciferase reporter gene. GC levels are normalized to the maximum observed value. (Figure 8B) Purified time-course vectors were used to transduce Huh7 cells. Transduction efficiency (luminescence units per GC added to target cells) was measured using multiple dilutions of purified time-course vector samples as in Figure 8B. Error bars represent the standard deviation of at least 10 technical replicates for each sample time point. Deamidation of AAV8 NG (Figure 9C) and non-NG (Figure 9D) sites in vectors collected 1, 2, and 5 days after transfection. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figures 10A-10B]Figure 10 illustrates the impact of asparagine stabilization on vector performance. Figure 10A shows the titers of wtAAV8 and +1 position mutant vectors produced by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). Titers are reported relative to the wtAAV8 control. Figure 10B shows the transduction efficiencies of the reported firefly luciferase-producing mutant AAV8 vectors compared to the wtAAV8 control. Transduction efficiencies were measured as in Figure 8B using crude vector material. Two-sample t-tests (*p<0.005) were performed to determine significance between wtAV8 and mutant transduction efficiencies for G264A / G515A and G264A / G541A. Figure 10C shows luciferase expression on day 14 of the study period in the liver region from C57BL / 6 mice intravenously injected with wtAAV8 or mutant vectors (n = 3-5), as measured by luciferase imaging and reported in total flow units. Figure 10D shows the titer and transduction efficiency of the reported firefly luciferase-producing multi-site AAV8 mutant vectors compared to the wtAAV8 control. All data are expressed as mean ± standard deviation. [Figures 11A-11C] Analysis of asparagine and glutamine deamidation in AAV9 capsid proteins. (Figure 11A) 1e11GC wtAAV9 was analyzed by 2D gel electrophoresis and stained with Sypro Ruby. Protein labels: A = VP1, B = VP2, C = VP3, D = chicken egg white conalbumin marker, E = turbonuclease marker. Isoelectric focusing was performed over a pI range of 6 to 10. (Figure 11B) Deamidation rates at specific asparagine and glutamine residues of interest are shown for AAV9 tryptic peptides purified by different methods. Bars indicating deamidation at asparagine residues with N+1 glycine are shaded. Residues determined to be at least 2% deamidated in at least one preparation analyzed were included. Data are presented as mean ± standard deviation. (FIG. 11C) The isoasparagine model of N512 is shown in the 2FoFc electron density map (PDB number: 3UX1) generated by unbiased refinement of the AAV9 crystal structure. The arrow indicates the electron density corresponding to the R group of residue N512. [Figures 11D-11F] Determining factors affecting AAV9 capsid deamidation. (Figure 11D) Two AAV9 preparations were incubated at 70°C for 3 or 7 days and (Figure 11F) exposed to pH 2 or pH 10 for 7 days to identify possible sources of deamidation not inherent to AAV capsid formation. Data are presented as mean ± standard deviation. (Figure 11F) Dot blot of vector processed as in Figure 11D using the B1 antibody (reactive with denatured capsids) to assess the integrity of the capsid structure. [Figures 11G-11H] Figure 11 illustrates an in vitro analysis of the impact of genetic deamidation on AAV9 vector performance. (Figure 11G) Titers of wtAAV9 and genetic deamidation mutant vectors produced by small-scale triple transfection in 293 cells measured by quantitative PCR (qPCR). Titers are reported relative to the wtAAV9 control. Highly deamidated NG sites (patterned bars), lowly deamidated sites (white bars), and highly variable sites (black bars) are displayed for wtAAV8 and negative controls. (Figure 11H) Transduction efficiencies of mutant AAV9 vectors producing firefly luciferase are reported relative to the wtAAV9 control. All data are expressed as mean ± standard deviation. [Figure 11A] Figures 11I-11K show the potency of AAV9 vectors over time. (Figure 11I) Vector production (DNAseI-resistant genome copies, GC) over a time course of triple-transfected HEK293 cells producing AAV9 vectors packaging a luciferase reporter gene. GC levels are normalized to the maximum observed value. (Figure 11J) Crude time course vectors were used to transduce Huh7 cells. (Figure 11K) Transduction efficiency of vectors harvested 1 day post-transfection versus 5 days post-transfection is shown for crude and purified vector samples. Transduction efficiency is expressed as luciferase activity / GC normalized to the value on day 1. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G] Same as above. [Figure 11H] Same as above. [Figure 11I] Same as above. [Figure 11J] Same as above. [Figure 11K] Same as above. [Figure 12A] Figures 12A-12B show characterization of PAV9.1 monoclonal antibodies and PAV9.1 epitope-based mutagenesis strategies. Figure 12A: PAV9.1 recognition of various AAV serotypes based on capture ELISA using native or denatured capsid proteins. Figure 12B: Alignment of AAV VP1 amino acid sequences (SEQ ID NOS: 10-19, top to bottom), with residues relevant to the PAV9.1 epitope in the black box. [Figure 12B] Same as above. [Figure 13] Figures 13A-D show cryo-EM reconstructions of AAV9 in complex with PAV9.1 Fab. Figure 13A: Representation of the molecular surface of the AAV9 capsid (fuchsia) bound to PAV9.1 Fab (blue with 3-fold axis projections reconstructed at 4.2 Å resolution). 3,022 particles are boxed, and Auto3dEM was used for electron microscopy reconstruction. Figure 13B: Representation of a cross section of the AAV9-PAV9.1 complex. Figure 13C: Pseudo-atomic model of the AAV9-PAV9.1 trimer embedded in the density obtained from the cryo-reconstruction. VP3 monomers are shown in green, gray, and cyan. Spheres represent binding residues. A single PAV9.1 Fab is depicted, with the heavy chain in indigo and the light chain in red. Figure 13D: Two-dimensional "roadmap" of residues involved in PAV9.1 binding. [Figure 14A]Figures 14A-14E show the effect of epitope mutations on the EC50 of PAV9.1 mAb for AAV9. A capsid capture ELISA for AAV9 was used to analyze and generate binding curves for PAV9.1. Figures 14A-14E show the following: 586-590 swap mutation (Figure 14A), 494-498 mutation (Figure 14B), 586-590 point mutation (Figure 14C), AAV9.TQAAA and AAV9.SAQAN single and combination mutations (Figure 14D), and AAV9.TQAAA and AAV9.SAQAA single and combination mutations (Figure 14E). Absorbance was normalized to the maximum absorbance of each capsid. The line of best fit and EC50 were determined using the dose-response function in Prism. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 15A] Figures 15A-15K characterize the effect of PAV9.1 epitope mutations on in vivo vector transduction and effective PAV9.1 mAb neutralization titers. Figure 15A: Transduction efficiency of PAV9.1 capsid mutations compared to AAV9.WT in HEK293 cells. Significance was determined using a two-tailed, one-sample t-test, comparing the percent transduction of each mutation to that of AAV9.WT (defined as 100%). P values ​​are indicated as p*<0.05, p***<0.001. Figures 15B-15k: The neutralization titer of PAV9.1 is determined when HAV293 cells are transduced with AAV9.WT.CMV.LacZ (Figure 15B), AAV9.AAQAA (Figure 15C), AAV9.QQNAA (Figure 15D), AAV9.SSNTA (Figure 15E), AAV9.RGNRQ (Figure 15F), AAV9.RGHRE (Figure 15G), AAV9.TQAAA (Figure 15H), AAV9.AANNN (Figure 15I), AAV9.SAQAN (Figure 15J), or AAV9.SAQAA (Figure 15K). The neutralization titer was defined as the dilution before which a transduction level 50% or higher than the vector without mAb (levels measured in relative light units) could be achieved. All data are reported as mean ± SD. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above. [Figure 15H] Same as above. [Figure 15I] Same as above. [Figure 15J] Same as above. [Figure 15K] Same as above. [Figure 16] Figure 16 shows the correlation between PAV9.1 EC50 and neutralization titer for a panel of AAV9 mutations. The fold reduction in PAV9.1 neutralization titer for each mutation was calculated compared to the PAV9.1 neutralization titer for AAV9.WT. Data were plotted on a logarithmic scale against the fold increase in PAV9.1 EC50 for each mutation compared to the PAV9.1 EC50 for AAV9.WT on a linear scale (semi-log plot). The best-fit semi-log line was determined using GraphPad Prism; R2 = 0.8474. [Figure 17A] Figures 17A-17G show in vivo analysis of AAV9 PAV9.1 mutant vectors. C57BL / 6 mice received intravenous injections of either AAV9.CMV.LacZ (WT or mutant; n = 3) at 1e11 GC per mouse (Figures 17A-17C) or 1e12 GC per mouse (Figures 17D-17F). Mice were sacrificed on day 14, and tissues were collected for biodistribution analysis (Figures 17A and 17D) using Taqman qPCR. Values ​​are reported as mean ± SD. Liver (Figures 17B and 17E), heart (Figures 17C and 17F), and muscle (Figure 17G) were also collected for β-gal histochemistry to determine enzyme activity. Representative 10x images are shown; scale bar = 200 μm. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above. [Figure 17F] Same as above. [Figure 17G] Same as above. [Figure 18A] Figures 18A-18D show the effect of epitope mutations on the EC50 of injected mouse plasma for AAV9. Using capsid capture ELISA, plasma was analyzed on day 56 from mice receiving intravenous infusions of either AAV9.WT or the AAV9 PAV9.1 mutant-binding wtAAV9.LSP.hFIX at 7.5e8 GC / mouse (Figure 18A) or 7.5e9 GC / mouse (Figure 18B). Absorbance was normalized to the maximum absorbance achieved with each capsid. The line of best fit and EC50 were determined using the dose-response function in Prism. Each graph corresponds to a single animal. EC50 values ​​were collected for 7.5e8 GC / mouse (Figure 18C) or 7.5e9 GC / mouse (Figure 18D) to determine the average value for each mutation. A two-tailed, one-sample t-test was used to determine whether the plasma EC50 of each mutant was significantly different compared to the plasma EC50 of AAV9.WT (defined as 1). A Bonferroni correction was applied to control for type 1 error. P values ​​are expressed as follows: ** = p < 0.05, ** = p < 0.01, *** = p < 0.001. EC50 data are reported as mean ± SD. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D] Same as above. [Figure 19A]Figures 19A-19D show the effect of epitope mutations on the EC50 of NHP polyclonal serum for AAV9. Using a capsid capture ELISA, serum from NHPs treated with (Figure 19A) AAV9.WT or hu68.WT vectors or untreated NHPs that were AAV9 NAb(+) for (Figure 19B) AAV9.WT or AAV9 PAV9.1 mutant binding was analyzed. Absorbance was normalized to the maximum absorbance achieved with each capsid. The optimal EC50 was determined using the dose-response function in Prism. Each graph corresponds to a single animal. EC50 values ​​for vector-treated NHPs (Figure 19C) and untreated NAb(+) NHPs were pooled to determine the mean for each mutation (Figure 19D). A two-tailed, one-sample t-test was used to determine whether the plasma EC50 of each mutation was significantly different from the plasma EC50 of AAV9.WT (defined as 1). Bonferroni correction was applied to control for type 1 error. EC50 data are reported as mean ± SD. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above. [Figure 20A] Figures 20A-20B show the effect of epitope mutations on the EC50 of human donor polyclonal sera for AAV9. Figure 20A: Using a capsid capture ELISA, serum from untreated human donors who were AAV9 NAb(+) was analyzed for binding of AAV9.WT or the AAV9 PAV9.1 mutant. The dose-response function in Prism was used to determine the line of best fit and EC50. Each graph corresponds to a single donor. Figure 20B: EC50 values ​​for NAb(+) human donor sera were collected and the mean for each mutation was determined. Significance was determined using a two-tailed, one-sample t-test, comparing the EC50 of each mutation's plasma to the EC50 of AAV9.WT plasma (defined as 1). A Bonferroni correction was applied to control for type 1 error. EC50 data are reported as mean ± SD. [Figure 20B] Same as above. [Figure 21]Figures 21A-B show AAV8 in vitro titer and transduction data from 6-well plate-scale experiments, including the N57Q, N263Q, N385Q, N514Q, N540Q, N94Q, and N410Q mutations for AAV8. [Figure 22] Figures 22A-B show AAV9 in vitro titer and transduction data from 6-well plate-scale experiments, including the N57Q, N329Q, N452Q, N270Q, N409Q, N668Q, N94Q, N253Q, N663Q, and N704Q mutations for AAV9. [Figure 23] Figures 23A-23B provide in vivo transduction data for AAV8 and AAV9, respectively, in mice tested for liver expression in mice at day 14 (luciferase imaging). Figure 23A shows the AAV8 mutations N57Q, N263Q, and N385Q compared to wild-type AAV8. Figure 23B shows the AAV9 mutations N57Q, G58A, and G330A compared to wild-type AAV9. [Figure 24] Figures 24A-B illustrate the relative potency (GC) and transduction efficiency of the AAV9 double and triple mutations G330 / G453A, G330A / G513A, G453A / G513A, and G330 / G453A / G513A. Figure 24A compares the relative potency of the mutations to AAV9wt, and Figure 24B compares the relative transduction efficiency (luciferase / GC) of the mutations to AAV9wt. DETAILED DESCRIPTION OF THE INVENTION

[0018] Provided herein are recombinant adeno-associated viruses (rAAVs) having sequence and charge heterogeneity in each of the three capsid protein populations VP1, VP2, and VP3 found within the capsid of the recombinant AAV, as well as compositions comprising them. The invention provides novel rAAVs, as well as methods for reducing deamidation, and optionally other capsid monomer modifications.Further provided herein are modified rAAVs with reduced modifications, which are useful for providing rAAVs with capsids that maintain higher stability, potency, and / or purity.In certain embodiments, the rAAV is not AAVhu68.In certain embodiments, the rAAV is not AAV2.

[0019] In one embodiment, a composition comprises a mixed population of recombinant adeno-associated viruses (rAAVs), each of the rAAVs comprising (a) about 60 capsid vp1 proteins, vp2 proteins, and vp3 proteins, wherein the vp1, vp2, and vp3 proteins are selected from the group consisting of a heterogeneous population of vp1 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 proteins produced from a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence, a heterogeneous population of vp3 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 proteins produced from a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence, a heterogeneous population of vp1 proteins produced from a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence, a heterogeneous population of vp2 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 ...2 proteins produced from a nucleic acid sequence encoding a selected AAV vp1 amino acid sequence, a heterogeneous population of vp2 A composition is provided that includes: (a) a heterogeneous population of vp3 proteins produced from a nucleic acid sequence encoding a vp3 amino acid sequence, wherein the vp1, vp2, and vp3 proteins include a subpopulation with an amino acid modification comprising at least two highly deamidated asparagines (N) in asparagine-glycine pairs in the AAV capsid, and optionally further subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (b) a vector genome in the AAV capsid, wherein the vector genome comprises a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell. In certain embodiments, the composition is as described in this paragraph, except that the rAAV is not AAVhu68. As used herein, AAVhu68 is as defined in WO2018 / 160582. The predicted amino acid sequence of AAVu68 VP1 is reproduced in SEQ ID NO: 114, and the native nucleic acid sequence is provided in SEQ ID NO: 113. In certain embodiments, the composition is as described in this paragraph, except that the rAAV is not AAV2.

[0020] In certain embodiments, a mixed population of rAAVs results from a production system that uses a single AAV capsid nucleic acid sequence that encodes the predicted AAV VP1 amino acid sequence of one AAV type, however, the production and manufacturing process provides a heterogeneous population of the capsid proteins.

[0021] In certain embodiments, recombinant AAVs are provided that have mutant AAV8 capsids that have one or more improved properties compared to unmodified AAV8 capsids. Such improved properties may include, for example, increased titer and / or increased relative transduction efficiency compared to AAV8. In certain embodiments, the mutations may include AAV8 G264A / G515A (SEQ ID NO: 21), AAV8 G264A / G541A (SEQ ID NO: 23), AAV8 G515A / G541A (SEQ ID NO: 25), or AAV8 G264A / G515A / G541A (SEQ ID NO: 27). In certain embodiments, nucleic acid sequences encoding these mutant AAV8 capsids are provided. In certain embodiments, the nucleic acid sequence is provided in, for example, SEQ ID NO: 20 (AAV8 G264A / G515A), SEQ ID NO: 22 (AAV8 G264A / G541A), SEQ ID NO: 24 (AAV8 G515A / G541A), or SEQ ID NO: 26 (AAV8 G264A / G515A / G541A). In certain embodiments, the AAV8 mutations can be N499Q, N459Q, N305Q / N459Q, N305QN499Q, N459Q, N305Q / N459Q, N305q / N499Q, or N205Q, N459Q, or N305Q / N459Q, N499Q. In certain embodiments, these mutations are combined with the G264A / G541A mutation. In certain embodiments, the mutations are AAV8 G264A / G541A / N499Q (SEQ ID NO: 115), AAV8 G264A / G541A / N459Q (SEQ ID NO: 116), AAV8 G264A / G541A / N305Q / N459Q (SEQ ID NO: 117), AAV8 G264A / G 541A / N305Q / N499Q (SEQ ID NO: 118), G264A / G541A / N459Q / N499Q (SEQ ID NO: 119), or AAV8 G264A / G541A / N305Q / N459Q / N499Q (SEQ ID NO: 120). In other embodiments, the single mutations can be, or be selected from, e.g., AAV8N263A, AAV8N514A, AAV8N540A. In certain embodiments, other AAVs can be mutated to have these or corresponding N-G pair changes based on alignment with AAV8. Such AAVs can be clade E AAVs. See, for example, the AAV8 mutations described in Example 2 (SEQ ID NO: 9).

[0022] In certain embodiments, the AAV8 mutations avoid altering the N-G pair at positions N57, N94, N263, N305, G386, Q467, N479, and / or N653. In certain embodiments, the other AAV avoids mutations at the corresponding N positions, as determined based on alignment with AAV8, using AAV8 numbering as a reference.

[0023] In certain embodiments, recombinant AAVs are provided that have mutant AAV9 capsids that have one or more improved properties compared to unmodified AAV9 capsids. Such improved properties may include, for example, increased titer and / or increased relative transduction efficiency compared to AAV9. In certain embodiments, the mutant AAV9 capsid may include, for example, AAV9 G330 / G453A (SEQ ID NO: 29), AAV9 G330A / G513A (SEQ ID NO: 31), AAV9 G453A / G513A (SEQ ID NO: 33), and / or AAV9 G330 / G453A / G513A (SEQ ID NO: 35). In certain embodiments, nucleic acid sequences encoding these mutant AAV9 capsids are provided. In certain embodiments, nucleic acid sequences are provided in, for example, SEQ ID NO:28 (9G330AG453A), SEQ ID NO:30 (9G330AG513A), SEQ ID NO:32 (9G453AG513A), SEQ ID NO:34 (9G330AG453AG513A). In certain embodiments, other AAVs can be mutated to have these or corresponding NG pair changes based on alignment with AAV 9. Such AAVs can be clade F AAVs.

[0024] In certain embodiments, rAAVs having a Clade A, Clade B, Clade C, or Clade D mutant AAV capsid can be engineered to have N-G pair amino acid modifications corresponding to those identified above for Clade E and Clade F. In certain embodiments, the Clade A (e.g., AAV) mutation can include a mutation at position N303, N497, or N303 / N497 with reference to the numbering of SEQ ID NO: 1 (AAV1). In certain embodiments, the mutation is N497Q. In certain embodiments, the AAV3B mutation can include a mutation at position N302, N497, or N302 / N497 with reference to the numbering of SEQ ID NO: 2. In certain embodiments, the mutation is N497Q. In certain embodiments, the AAV5 mutation can include a mutation at position N302, N497, or N302 / N497 with reference to the numbering of SEQ ID NO: 3. In a particular embodiment, the mutation is N497Q.

[0025] Without intending to be bound by theory, mass spectrometry analysis revealed asparagine deamidation at several positions on the capsid as an explanation for the existence of multiple VP isoforms not previously described for AAV. Additionally, the distribution and extent of deamidation was consistent across several methods of vector purification, suggesting that this phenomenon occurs independently of vector processing. The functional significance of these deamidations was investigated by individually mutating several asparagines to aspartic acid. A subset of these mutations affected not only the efficiency of particle assembly but also the vector's ability to transduce target cells both in vitro and in vivo. De novo modeling of these deamidated residues into the AAV8 structure also revealed the structural basis for the existence of these deamidation events and demonstrated how the AAV8 capsid tolerates these changes in amino acid identity and properties. We provide a computational explanation for why. Virtually identical findings of deamination were observed in AAV9 and various additional AAVs. Thus, rAAV features a previously unknown AAV capsid structural isomerism.

[0026] In the study reported herein, we found that extensive asparagine and occasional glutamine deamidation is affected by 17 residues. The factors controlling AAV8 deamidation, primarily primary sequence and 3D structural constraints, are likely conserved across the AAV phylogeny, as all serotypes we have analyzed so far exhibit remarkably similar modification patterns. Thus, deamidation is a potentially important factor in the development of future AAV therapeutics.

[0027] Following this discovery, we were motivated to explore the functional consequences of AAV deamidation. The multimeric nature of AAV vector capsids, the extent and number of modified capsid residues, and the mosaic diversity in the resulting vector particle composition presented several special challenges for this analysis. The experimental repertoire capable of adequately parameterizing the impact of post-translational modifications (PTMs) in the context of simpler proteins has not been directly applied to AAV capsid analysis. For example, it would be impossible to purify or enrich preparations of specific deamidated vector species to test their function directly and in isolation.

[0028] Genetic substitution with aspartate is one approach that attempts to enforce approximation of the modification at a given site. Beyond the aforementioned differences between the distribution of position-specific modifications on capsid assembly with endogenous (mosaic) versus genetic (complete) deamidation, our data point to additional considerations for interpreting this data. For example, we observed a greater than 50-fold transduction loss with the N263D mutation compared to wtAAV8 (Figure 8B). This is surprising given the minimal change in aspartate content at this position due to genetic conversion; N263 is 99% deamidated in wtAAV8. One explanation for this difference is that the products of genetically encoded aspartate and asparagine deamidation are molecularly distinct (L-aspartate versus a presumed 3:1 mixture of L / D-isoaspartate:L / D-aspartate). Therefore, genetic approximation may be insufficient at some positions. Another residue, the highly conserved N57, also did not tolerate substitution with aspartate, but was deamidated to an average of 80% and 97% in AAV8 and AAV9, respectively (Figures 8B and 11). Here, the residual intact amide may buffer activity in wild-type preparations through a mosaic effect. We also detected the possibility of crosstalk with other asparagines confounding the analysis of N57, and the adjacent N66 was significantly deamidated when the amide at position 57 was mutagenically conserved (N57Q, G58A, and G58S for AAV8; N57Q and G58A for AAV9; data not shown). While this was the only case of crosstalk detected by mass spectrometry analysis of the mutations, it highlights another complexity in interpreting loss-of-function mutagenesis data.

[0029] With these caveats in mind, we developed evidence for the impact of deamidation through time-course and gain-of-function mutagenesis experiments. Our data are consistent with the role of a subset of NG sites in the loss of function associated with deamidation at very early time points. To our knowledge, this event has not been reported previously. Indeed, the specific experimental procedures we used to identify this decay were informed by the novel observation of very short half-life vector NG deamidation, and storing early samples in the refrigerator for even a day may diminish their distinction from later time point samples, given the rate of spontaneous deamidation we observed. While storage stability experiments comparing the activity of vector preparations over days or weeks after processing are routine in our laboratory and other manufacturing groups, these comparisons are mostly performed on vector material that is at least 7 days old, when most or all of the activity decay (and NG site deamidation) has completed. The data suggest that process interventions or modifications to obtain improved capsids may be necessary. highlights the opportunity for N-stabilizing mutagenic approaches. From a broader perspective, it is also important to consider the role of the "deamidation clock" in the natural biology of AAV, a phenomenon that is speculated to give the most recently translated viral particles in infected cells an advantage in the next round of infection.

[0030] Although we did not explore the mechanistic basis for NG deamidation-induced loss of function, several prominent possibilities exist. All NG motifs in AAV8 and AAV9 VP3 are located in surface HVR loops. In AAV8, NGs 514 and 540 are located near the 3-fold axis within a region known to play an important role in transduction through interactions with cellular receptors. While the AAV8 receptor binding site has not been fully investigated, the LamR receptor has been implicated in AAV8 transduction. These studies identify aa 491-557 as critical for these interactions. Functional investigations of the capsid identified residues in the AAV9 galactose-binding domain, thereby characterizing AAV9 receptor binding as superior to AAV8. Among these residues, we found that a single asparagine, N515, was deamidated at low levels (3%), while the other two asparagines in this domain, N272 and N470, were not deamidated. Thus, deamidation may affect galactose binding, but probably only to a small extent.

[0031] In summary, we have identified how AAV vector deamidation can affect transduction efficiency and demonstrated strategies to stabilize deamidation and improve vector performance. A major future goal will be to extend these findings to appropriate animal model systems to examine the impact of deamidation and the performance of our stabilized variants in more complex functional contexts. Potential implications for tissue tropism and capsid interactions with the immune system must be carefully evaluated. Because these complex effects would be extremely difficult to definitively determine for all deamidated residues in the capsid, it may be prudent to target a limited number of residues with high lot-to-lot variability for deamidation for stabilization by mutagenesis, as was successfully achieved for the variable AAV8 asparagines 459 and 499. Additionally, deamidation analysis of vector preparations using our mass spectrometry workflow may prove beneficial in achieving functional consistency across manufactured lots of AAV gene therapy products.

[0032] "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle containing two elements: an AAV capsid and a vector genome containing at least a non-AAV coding sequence packaged within the AAV capsid. Unless otherwise specified, this term can be used interchangeably with the phrase "rAAV vector." rAAV lacks any functional AAV rep gene or functional AAV cap gene and is therefore unable to produce progeny, making it a "replication-defective virus" or "viral vector." In certain embodiments, the only AAV sequence is an AAV inverted terminal repeat (ITR), typically located at the 5' and 3' extreme ends of the vector genome to allow the genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0033] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside the rAAV capsid that forms the viral particle. Such nucleic acid sequences include AAV inverted terminal repeats (ITRs). In the examples herein, the vector genome comprises, at least from 5' to 3', the AAV 5' ITR, a coding sequence, and the AAV 3' ITR. ITRs from AAV2, a source AAV different from the capsid, or other than full-length ITRs can be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs can be used. Additionally, the vector genome includes regulatory sequences that direct the expression of gene products. Suitable components of a vector genome are discussed in more detail herein.

[0034] rAAV consists of AAV capsid and vector genome. AAV capsid is a collection of a heterogeneous population of vp1, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein, when referring to vp capsid proteins, the term "heterogeneous" or any grammatical variant thereof refers to a collection of non-identical elements, for example, vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0035] As used herein, the term "heterologous" in reference to the vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1, vp2, and vp3 proteins, each with modifications from predicted amino acid residues. These subpopulations contain at least specific deamidated asparagine (N or Asn) residues. For example, specific subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where deamidation results in amino acid changes and any other modifications.

[0036] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that share at least one defined common characteristic and consist of at least one group member but fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins, unless otherwise specified, is at least one (1) vp1 protein but fewer than all vp1 proteins in an assembled AAV capsid. A "subpopulation" of vp3 proteins, unless otherwise specified, can be one (1) vp3 protein but fewer than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subpopulation of vp proteins, vp2 proteins can be a separate subpopulation of vp proteins, and vp3 can be a further subpopulation of vp proteins in an assembled AAV capsid. In another example, the vp1, vp2, and vp3 proteins can include subpopulations with different modifications, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., asparagine-glycine pairs.

[0037] Unless otherwise specified, highly deamidated refers to at least 45% deamidation, at least 50% deamidation, at least 60% deamidation, at least 65% deamidation, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or up to about 100% deamidation at a reference amino acid position compared to the predicted amino acid sequence at the reference amino acid position (e.g., At least 80% of the asparagine at amino acid 57 based on the numbering of SEQ ID NO: 1 [AAV1], 2 [AAV3B], 4 [AAV7], 5 [AAVrh32.33], 6 [AAV8], 7 [AAV9], 9 [AAV8 triple], or 111 [AAVhu37], or amino acid 56 based on the numbering of SEQ ID NO: 3 [AAV5], may be deamidated based on the total vp1 protein, and may be deamidated based on the total vp1, vp2, and vp3 proteins. Such percentages may be determined using 2D gels, mass spectrometry techniques, or other suitable techniques.

[0038] As used herein, a "deamidated" AAV is one in which one or more amino acid residues have been derivatized to a residue other than that encoded for it in the corresponding nucleic acid sequence.

[0039] Without intending to be bound by theory, it is believed that the vp protein in the AAV capsid Deamidation of at least the highly deamidated residues in VP1 is thought to be nonenzymatic in nature and is triggered by functional groups within the capsid protein that deamidate selected asparagine and, to a lesser extent, glutamine residues. The efficient capsid assembly of the majority of deamidated VP1 proteins indicates that these events occur after capsid assembly, or that deamidation in individual monomers (VP1, VP2, or VP3) is structurally well tolerated and largely does not affect assembly kinetics. In general, extensive deamidation in the VP1-unique (VP1-u) region (~aa1-137), which is thought to be internally located prior to cell entry, suggests that VP deamidation may occur prior to capsid assembly. N-deamidation may occur by nucleophilic attack on the side-chain amide carbon atom of Asn via the backbone nitrogen atom of its C-terminal residue. An intermediate ring-closing succinimide residue is thought to form. The succinimide residue then undergoes rapid hydrolysis to yield the end products aspartic acid (Asp) or isoaspartic acid (IsoAsp). Thus, in certain embodiments, deamidation of asparagine (N or Asn) yields Asp or IsoAsp, which can be interconverted via a succinimide intermediate, for example, as exemplified below. [ka]

[0040] As provided herein, each deamidated N in VP1, VP2, or VP3 can independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and / or an interconverting blend of Asp and isoAsp, or a combination thereof. Any suitable ratio of α- and isoaspartic acid can be present. For example, in certain embodiments, the ratio can be 10:1 to 1:10 asparagine to isoasparagine, about 50:50 asparagine:isoasparagine, or about 1:3 asparagine:isoasparagine, or another selected ratio.

[0041] In certain embodiments, one or more glutamines (Q) can be derivatized (deamidated) to glutamic acid (Glu), i.e., α-glutamic acid, γ-glutamic acid (Glu), or a blend of α- and γ-glutamic acid, which can be interconverted via a common glutarimide intermediate. Any suitable ratio of α- and γ-glutamic acid can be present. For example, in certain embodiments, the ratio can be 10:1 to 1:10 α:γ, about 50:50 α:γ, or about 1:3 α:γ, or another selected ratio. [ka]

[0042] Thus, the rAAV contains subpopulations of vp1, vp2, and / or vp3 proteins in the rAAV capsid that have deamidated amino acids, including at least one subpopulation that contains at least one highly deamidated asparagine. Additionally, other modifications may include isomerization at specifically selected aspartic acid (D or Asp) residue positions. In yet other embodiments, modifications may include amidation at Asp positions.

[0043] In certain embodiments, AAV capsids comprise subpopulations of vp1, vp2, and vp3 having at least 4 to at least about 25 deamidated amino acid residue positions, of which at least 1-10% are deamidated relative to the encoded amino acid sequence of the vp protein. The majority of these may be N residues. However, Q residues may be deamidated.

[0044] In certain embodiments, the rAAV has an AAV capsid with vp1, vp2, and vp3 proteins with subpopulations containing combinations of two, three, four, or more deamidated residues at the positions indicated in the tables provided in the Examples and incorporated herein by reference. The deamidation of rAAV can be determined using 2D gel electrophoresis, mass spectrometry, and / or protein modeling techniques. Online chromatography was performed using an Acclaim PepMap column coupled to a Q Exactive HF with a NanoFlex source (Thermo Fisher Scientific) and a Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific). MS data were acquired using the data-dependent Top-20 method on the Q Exactive HF, which dynamically selects the most abundant, unsequenced precursor ions from the survey scan (m / z 200–2000). Sequencing was performed via higher-energy collisional dissociation fragmentation with a target value of 1e5 ions determined by predictive autoincrement control, allowing precursor isolation over a 4 m / z window. Survey scans were acquired at a resolution of 120,000 at m / z 200. HCD spectral resolution was set to 30,000 at m / z 200 with a maximum ion injection time of 50 ms and a normalized collision energy of 30. The S-lens RF level was set to 50 to optimize transmission of the m / z region occupied by peptides from the digest. Precursor ions could be single, unassigned, or excluded from the fragmentation selection with six or more charge states. BioPharma Finder 1.0 software (Thermo Fischer Scientific) was used to analyze the acquired data. For peptide mapping, a single-entry protein FASTA database was created with carbamidomethylation as the fixed modification and oxidation, deamidation, and phosphorylation as variable modifications. Searches are performed using a mass spectrometric (MS / MS) parameter set, 10 ppm mass accuracy, high protease specificity, and a confidence level of 0.8 for MS / MS spectra. Examples of suitable proteases include trypsin or chymotrypsin. Mass spectrometric identification of deamidated peptides is relatively straightforward, as deamidation adds 0.984 Da (the mass difference between the -OH and -NH groups) to the mass of the intact molecule. The deamidation rate for a particular peptide is determined by dividing the mass area of ​​the deamidated peptide by the sum of the areas of the deamidated and native peptides. Considering the number of possible deamidation sites, isotopic species deamidated at different sites may comigrate in a single peak. Therefore, fragment ions derived from peptides with multiple potential deamidation sites can be used to identify or distinguish between multiple deamidation sites. In these cases, the relative intensities within the observed isotopic pattern can be used to specifically determine the relative abundance of different deamidated peptide isomers. This method assumes that the fragmentation efficiency for all isomeric species is the same and is independent of the deamidation site. Those skilled in the art will understand that some variations of these exemplary methods can be used. For example, suitable mass analyzers can include quadruple flight mass analyzers (QTOF), such as the Waters Xevo or Agilent 6530, or orbitrap devices such as the Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Suitable liquid chromatography systems can include, for example, the Acquity UPLC system from Waters or Agilent systems (1100 or 1200 series). Suitable data analysis software can include, for example, MassLynx (Waters), Pinpoint and Pepfinder (Thermo Fisher Scientific), Mascot (Matrix Science), Peaks DB (Bioinformatics Solutions).Further techniques may be described, for example, in X. Jin et al., Hu Gene Therapy Methods, Vol. 28, No. 5, pp. 255-267, published online June 16, 2017.

[0045] In addition to deamidation, other modifications may occur that do not convert one amino acid into a different amino acid residue, and such modifications may include acetylated residues, isomerization, phosphorylation, or oxidation.

[0046] Modulation of deamidation: In certain embodiments, AAV is modified to change the glycine of an asparagine-glycine pair to reduce deamidation. In other embodiments, the asparagine is changed to a different amino acid, such as glutamine, which deamidates at a slower rate, or to an amino acid lacking an amide group (e.g., glutamine and asparagine contain an amide group), and / or an amino acid lacking an amine group (e.g., lysine, arginine, and histidine contain an amine group). As used herein, an amino acid lacking an amide or amine side group refers to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, phenylalanine, tyrosine, or tryptophan, and / or proline. The described modifications can be present in one, two, or three of the asparagine-glycine pairs found in the encoded AAV amino acid sequence. In certain embodiments, such modifications are not present in all four asparagine-glycine pairs. Provided herein are methods for reducing deamidation of AAV and / or engineered AAV variants with a lower deamidation rate. Additionally or alternatively, one or more other amide amino acids may be changed to non-amide amino acids to reduce AAV deamidation. In certain embodiments, the mutant AAV capsids described herein contain a mutation in an asparagine-glycine pair, changing glycine to alanine or serine. The mutant AAV capsid may contain one, two, or three mutations where the reference AAV naturally contains four NG pairs. In certain embodiments, the AAV capsid may contain one, two, three, or four such mutations where the reference AAV naturally contains five NG pairs. In certain implementations, In some embodiments, the mutant AAV capsid contains only a single mutation in the NG pair. In certain embodiments, the mutant AAV capsid contains mutations in two different NG pairs. In certain embodiments, the mutant AAV capsid contains two different NG pairs containing mutations, which are located at structurally distinct positions in the AAV capsid. In certain embodiments, the mutations are not in the VP1 unique region. In certain embodiments, one of the mutations is in the VP1 unique region. Optionally, the mutant AAV capsid does not contain a modification in the NG pair, but contains a mutation to minimize or eliminate deamidation in one or more asparagines or glutamines located outside the NG pair.

[0047] In certain embodiments, a method for increasing the potency of an rAAV vector is provided, comprising engineering an AAV capsid to eliminate one or more NGs in a wild-type AAV capsid. In certain embodiments, the coding sequence for the "G" of "NG" is engineered to encode a different amino acid. In the specific example below, an "S" or an "A" is substituted. However, other suitable amino acid coding sequences may be selected. For example, see the table below for the coding sequence of at least one of the following positions based on the AAV8 numbering: N57+1, N263+1, N385+1, N514+1, and N540+1 are modified. In certain embodiments, AAV8 mutations avoid altering the NG pair at positions N57, N94, N263, N305, Q467, N479, and / or N653. In certain embodiments, other AAVs avoid mutations at the corresponding N positions, as determined based on alignment with AAV8 using the AAV8 numbering as a reference.

[0048] These amino acid modifications can be made by conventional genetic engineering techniques. For example, a nucleic acid sequence containing a modified AAV vp codon can be generated, in which one to three of the codons encoding glycine in an asparagine-glycine pair are modified to encode an amino acid other than glycine. In certain embodiments, a nucleic acid sequence containing a modified asparagine codon can have one to three of the asparagine-glycine pairs engineered so that the modified codon encodes an amino acid other than asparagine. Each modified codon can encode a different amino acid. Alternatively, one or more of the altered codons can encode the same amino acid. In certain embodiments, these modified AAV nucleic acid sequences can be used to generate mutant rAAVs with capsids that have less deamidation than native capsids. Such mutant rAAVs can have reduced immunogenicity and / or increased stability during storage, particularly storage in suspension form.

[0049] Also provided herein are nucleic acid sequences encoding AAV capsids with reduced deamidation. Designing nucleic acid sequences encoding this AAV capsid, including DNA (genomic or cDNA) or RNA (e.g., mRNA), is within the skill of the art. Such nucleic acid sequences can be codon-optimized for expression in a selected system (i.e., cell type) and can be designed by a variety of methods. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, CA). One codon optimization method is described, for example, in U.S. International Patent Publication No. WO2015 / 012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication Nos. 2014 / 0032186 and 2006 / 0136184. Preferably, the entire open reading frame (ORF) of the product is modified. However, in some embodiments, only a fragment of the ORF may be modified. Using one of these methods, a frequency can be applied to any given polypeptide sequence to generate nucleic acid fragments of codon-optimized coding regions that encode the polypeptide. Several options are available for performing the actual changes to the codons or synthesizing the codon-optimized coding regions designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biology procedures well known to those skilled in the art. In one approach, each 80-90 nucleotide A series of complementary oligonucleotide pairs spanning the length of the oligonucleotide and the length of the desired sequence are synthesized by standard methods. These oligonucleotide pairs are synthesized to form 80-90 base pair double-stranded fragments containing cohesive termini when annealed; for example, each oligonucleotide in a pair is synthesized so that it extends 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region complementary to the other oligonucleotide in the pair. The single-stranded end of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are annealed, and then approximately 5-6 of these double-stranded fragments are annealed together via the cohesive single-stranded ends, which are then ligated together and cloned into a standard bacterial cloning vector, such as the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs are prepared, consisting of five or six 80-90 base pair fragments ligated together, i.e., approximately 500 base pair fragments, so that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be readily apparent to those skilled in the art. Furthermore, gene synthesis is readily available commercially.

[0050] In certain embodiments, AAV capsids are provided that have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) that contain multiple highly deamidated "NG" positions. In certain embodiments, the highly deamidated positions are at the positions shown below, with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified to remove the referenced "NG" and the mutated "NG" is engineered into another position.

[0051] In certain embodiments, AAV1 is characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid as determined using mass spectrometry.

[0052] In certain embodiments, the AAV capsid is modified at one or more of the following positions, in the ranges provided below, as determined using mass spectrometry: Suitable modifications include those described in the above paragraph, labeled by deamidation, and are incorporated herein.

[0053] In certain embodiments, one or more of the following positions, or glycines following an N, are modified as described herein. In certain embodiments, AAV1 mutants are constructed in which the glycines following the N at positions 57, 383, 512, and / or 718 are preserved (i.e., left unmodified). In certain embodiments, the N at the four positions indicated above is preserved in the native sequence. Residue numbers are based on the published AAV1 VP1, reproduced in SEQ ID NO: 1.

[0054] In certain embodiments, the artificial NG is introduced at a position different from one of the positions shown below.

[0055] Residue numbers are based on the published AAV1 sequence reproduced in SEQ ID NO:1. [Table 1]

[0056] In certain embodiments, AAV3B capsids are characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid as determined using mass spectrometry. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled for deamidation and are incorporated herein. In certain embodiments, one or more of the following positions, or glycines following N, are modified as described herein. In certain embodiments, AAV3 mutants are constructed in which the glycines following N at positions 57, 383, 512, and / or 718 are preserved (i.e., left unmodified). In certain embodiments, the N residues at the four positions indicated above are conserved in the native sequence. Residue numbers are based on the published AAV3B VP1, reproduced in SEQ ID NO: 2. In certain embodiments, an artificial N residue is introduced at a position different from one of the positions shown below. In certain embodiments, the capsid is modified to reduce the "N" or "Q" at a position other than the "NG" pair. Residue numbers are based on the published AAV3B sequence reproduced in SEQ ID NO: 2. . [Table 2]

[0057] In certain embodiments, the AAV5 capsid is characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid as determined using mass spectrometry. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled with deamidation and are incorporated herein. In certain embodiments, one or more of the following positions, or the glycine following an N, are modified as described herein. In certain embodiments, an artificial N-G is introduced at a position other than one of the positions shown below. In certain embodiments, the capsid is modified to reduce an "N" or a "Q" at a position other than the "N-G" pair. Residue numbers are based on the published AAV5 sequence reproduced in SEQ ID NO: 3. [Table 3]

[0058] In certain embodiments, the AAV7 capsid is characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid as determined using mass spectrometry. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled with deamidation and are incorporated herein. In certain embodiments, one or more of the following positions, or a glycine following an N, are modified as described herein. In certain embodiments, an artificial N-G is introduced at a position other than one of the positions shown below. In certain embodiments, the capsid is modified to reduce an "N" or a "Q" at a position other than the "N-G" pair. Residue numbers are based on the published AAV7 sequence reproduced in SEQ ID NO:4. [Table 4]

[0059] In certain embodiments, AAVrh32.33 capsids are characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid, as determined using mass spectrometry. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled with deamidation and are incorporated herein. In certain embodiments, one or more of the following positions, or the glycine following an N, are modified as described herein. In certain embodiments, an artificial N-G is introduced at a position other than one of the positions shown below. In certain embodiments, the capsid is modified to reduce an "N" or a "Q" at a position other than the "N-G" pair. Residue numbers are based on the published AAVrh32.33 sequence reproduced in SEQ ID NO:5. [Table 5]

[0060] In certain embodiments, the AAV8 capsid is characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled for deamidation and are incorporated herein. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. In certain embodiments, one or more of the following positions, or glycines following an N, are modified as described herein. In certain embodiments, an artificial N-G is introduced at a position different from one of the positions shown below. In certain embodiments, an artificial N-G is introduced at a position different from one of the positions shown below. In certain embodiments, one or more of the following positions, or glycines following an N, are modified as described herein. For example, in certain embodiments, G can be modified to S or A at, for example, positions 58, 67, 95, 216, 264, 386, 411, 460, 500, 515, or 541. When NG57 / 58 is modified to NS57 / 58 or NA57 / 58, a significant decrease in deamidation is observed. However, in certain embodiments, when NG is modified to NS or NA, an increase in deamidation is observed. In certain embodiments, the N of the NG pair is modified to Q while retaining G. In certain embodiments, both amino acids of the NG pair are modified. In certain embodiments, N385Q results in a significant decrease in deamidation at that position. In certain embodiments, N499Q results in a significant increase in deamidation at that position. In certain embodiments, the NG mutation is made in the pair located at N263 (e.g., N263A). In certain embodiments, an NG mutation is made in the pair located at N514 (e.g., N514A). In certain embodiments, an NG mutation is made in the pair located at N540 (e.g., N540A). In certain embodiments, an AAV mutation is engineered that includes multiple mutations and at least one of the mutations at these positions. In certain embodiments, no mutation is made at position N57. In certain embodiments, no mutation is made at position N94.In certain embodiments, no mutation is made at position N305. In certain embodiments, the mutation is made at position G386. In certain embodiments, no mutation is made at position Q467. In certain embodiments, no mutation is made at position N479. In certain embodiments, no mutation is made at position N653. In certain embodiments, the capsid is modified to reduce an "N" or a "Q" at a position other than the "NG" pair. Residue numbers are based on the published AAV8 sequence reproduced in SEQ ID NO:6. [Table 6-1] [Table 6-2]

[0061] In certain embodiments, the mutations may include AAV8 G264A / G515A (SEQ ID NO:21), AAV8G264A / G541A (SEQ ID NO:23), AAV8G515A / G541A (SEQ ID NO:25), or AAV8 G264A / G515A / G541A (SEQ ID NO:27). In certain embodiments, nucleic acid sequences encoding these mutant AAV8 capsids are provided. In certain embodiments, the nucleic acid sequences are provided in, for example, SEQ ID NO:20 (AAV8 G264A / G515A), SEQ ID NO:22 (AAV8G264A / G541A), SEQ ID NO:24 (AAV8G515A / G541A), or SEQ ID NO:26 (AAV8 G264A / G515A / G541A). In certain embodiments, the AAV8 mutations can be N499Q, N459Q, N305Q / N459Q, N305QN499Q, N459Q, N305Q / N459Q, N305q / N499Q, or N205Q, N459Q, or N305Q / N459Q, N499Q. In certain embodiments, these mutations are combined with G264A / G541A mutations. In specific embodiments, the mutations are AAV8 G264A / G541A / N499Q (SEQ ID NO: 115), AAV8 G264A / G541A / N459Q (SEQ ID NO: 116), AAV8 G264A / G541A / N305Q / N459Q (SEQ ID NO: 117), AAV8 G264A / G541A / N305Q / N499Q (SEQ ID NO: 118), G264A / G541A / N459Q / N499Q (SEQ ID NO: 119), or AAV8 G264A / G541A / N305Q / N459Q / N499Q (SEQ ID NO: 120). Nucleic acid sequences encoding these AAV8 mutations are also encompassed.

[0062] In certain embodiments, AAV9 capsids are characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in the table below, based on the total amount of VP protein in the capsid, as determined using mass spectrometry. In certain embodiments, the AAV capsid is modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry. Suitable modifications include those described in the paragraph above that are labeled for deamidation and are incorporated herein. In certain embodiments, one or more of the following positions, or the glycine following an N, are modified as described herein. In certain embodiments, the N214 / G215 position encoded by AAV9 capsids is modified to N214Q, which is observed to have significantly increased deamidation. In certain implementations, In certain embodiments, an NG mutation is made in the pair located at N452 (e.g., N452A). In certain embodiments, no mutation is made at position N57. In certain embodiments, AAV mutations are engineered that include multiple mutations and at least one of the mutations at these positions. In certain embodiments, an artificial NG is introduced at a position other than one of the positions shown below. In certain embodiments, the capsid is modified to reduce the "N" or "Q" at a position other than the "NG" pair. Residue numbers are based on the published AAV9 sequence reproduced in SEQ ID NO:7. [Table 7]

[0063] Additionally or alternatively, AAVhu37 capsids comprise a heterogeneous population of vp1 proteins that are the product of nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:36, a heterogeneous population of vp2 proteins that are the product of nucleic acid sequences encoding the amino acid sequence from about amino acids 138 to 738 of SEQ ID NO:36, and a heterogeneous population of vp3 proteins that are the product of nucleic acid sequences encoding at least amino acids 204 to 738 of SEQ ID NO:36, wherein the vp1, vp2, and vp3 proteins comprise subpopulations with amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:36, and optionally further subpopulations comprising other deamidated amino acids, where deamidation results in an amino acid change. AAVhu37 is characterized by having highly deamidated residues at, for example, positions N57, N263, N385, and / or N514, based on the numbering of AAVhu37 VP1 (SEQ ID NO:36).

[0064] Deamidation has been observed at other residues, as shown in the table below and in the Examples. In certain embodiments, AAVhu37 capsids are modified at one or more of the following positions, within the ranges provided below, as determined using mass spectrometry with trypsin enzyme. In certain embodiments, one or more of the following positions, or glycines following an N, are modified as described herein. For example, in certain embodiments, a G can be modified to an S or an A, e.g., at positions 58, 264, 386, or 515. In one embodiment, an AAVhu37 capsid is modified from positions N57 / G58 to N57Q or G58A, resulting in a capsid with reduced deamidation at this position. In another embodiment, N57 / G58 is modified to NS57 / 58 or NA57 / 58. However, in certain embodiments, increased deamidation is observed when an NG is modified to an NS or NA. In certain embodiments, the N of the NG pair is modified to a Q while retaining the G. In certain embodiments, both amino acids of the N-G pair are modified. In certain embodiments, N385Q results in a significant reduction in deamidation at that position. In certain embodiments, N499Q results in a significant increase in deamidation at that position.

[0065] In certain embodiments, AAVhu37 may be deamidated at these or other residues, e.g., typically less than 10%, and / or methylated (e.g., at ∼R487) (typically less than 5% at a given residue, more typically less than 1%), isomerized (e.g., at D97) (typically less than 5% at a given residue, more typically less than 1%), phosphorylated (e.g., if present, about 10 to about 60%, or about 10 to about 30%, or In some embodiments, the amino acid sequence of the kynurenine amino acid sequence is 1 to 3, and the amino acid sequence of the kynurenine amino acid sequence is 2 to 3. The amino acid sequence of the kynurenine amino acid sequence is 1 to 3. The amino acid sequence of the kynurenine amino acid sequence is 1 to 3. The amino acid sequence of the kynurenine amino acid sequence is 1 to 3. The amino acid sequence of the kynurenine amino acid sequence is 1 to 3. The amino acid sequence of the kynurenine amino acid sequence is 1 to 3. [Table 8]

[0066] Still other positions may have these or other modifications (e.g., acetylation or further deamidation). In certain embodiments, the nucleic acid sequence encoding the AAVhu37 vp1 capsid protein is provided in SEQ ID NO:37. In other embodiments, a nucleic acid sequence that is 70% to 99.9% identical to SEQ ID NO:37 may be selected to express the AAVhu37 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99% to 99.9% identical to SEQ ID NO:37. However, other nucleic acid sequences that encode the amino acid sequence of SEQ ID NO:36 may be selected for use in producing rAAVhu37 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 37, or a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 37, and encodes SEQ ID NO: 36. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 37, or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 37 from about nt 412 to about nt 2214 of SEQ ID NO: 37, and encodes the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 36. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the nucleic acid sequence from about nt 610 to about nt 2214 of SEQ ID NO: 37, or to at least nt of SEQ ID NO: 37, and encodes the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 36. See EP 2 345 731 B1 and SEQ ID NO: 88 therein, which are incorporated by reference.

[0067] As used herein, "encoded amino acid sequence" refers to predicted amino acids based on translation of known DNA codons of a reference nucleic acid sequence that are translated into amino acids. The table below illustrates DNA codons and the 20 common amino acids, showing both the one-letter code (SLC) and the three-letter code (3LC). [Table 9]

[0068] rAAV vector As described above, the novel AAV sequences and proteins are useful for producing rAAV, and are also useful for recombinant AAV vectors, which can be antisense delivery vectors, gene therapy vectors, or vaccine vectors. In addition, the engineered AAV capsids described herein can be used to engineer rAAV vectors for the delivery of several suitable nucleic acid molecules to target cells and tissues.

[0069] The genomic sequence packaged in an AAV capsid and delivered to a host cell typically consists of at least a transgene, its regulatory sequence, and AAV inverted terminal repeats (ITRs). Both single-stranded AAV and self-complementary (sc) AAV are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory elements in a manner that allows the transcription, translation, and / or expression of the transgene in the cells of the target tissue.

[0070] The AAV sequence of the vector typically contains cis-acting 5' and 3' inverted terminal repeats (see, e.g., BJ Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modifications of these sequences are tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and K. Fisher et al., J. Virol., 70:520-532 (1996)). One example of such a molecule utilized in the present invention is a "cis-acting" plasmid containing a selected transgene sequence and associated regulatory elements flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are derived from an AAV other than the one providing the capsid. In one embodiment, the ITR sequences are from AAV2. A shortened version of the 5' ITR, termed ΔITR, has been described, in which the D sequence and terminal degradation site (trs) are deleted. In other embodiments, full-length AAV 5' and 3' ITRs are used. However, ITRs from other AAV sources may also be selected. If the ITRs are from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. However, other configurations of these elements may also be appropriate.

[0071] In addition to the key elements described above for recombinant AAV vectors, the vectors also contain the necessary conventional control elements operably linked to the transgene in a manner that allows for its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that flank the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0072] Regulatory control elements typically include promoter sequences located as part of expression control sequences, for example, between a selected 5'ITR sequence and a coding sequence. Constitutive promoters, regulatable promoters (see, for example, WO2011 / 126808 and WO2013 / 04943), tissue-specific promoters, or promoters that respond to physiological cues can be used in the vectors described herein. The promoter may be selected from different sources, for example, the human cytomegalovirus (CMV) immediate early enhancer / promoter, the SV40 immediate early enhancer / promoter, the JC polyomavirus promoter, the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, the herpes simplex virus (HSV-1) latency-associated promoter (LAP), the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, the neuron-specific promoter (NSE), the platelet-derived growth factor (PDGF) promoter, hSYN, the melanin-concentrating hormone (MCH) promoter, CBA, the matrix metalloprotein promoter (MPP), and the chicken beta-actin promoter. In addition to the promoter, the vector may include one or more other appropriate transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, such as WPRE, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, optionally, sequences that enhance secretion of the encoded product. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those appropriate for the desired target tissue indication. In one embodiment, the expression cassette includes one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or different from each other. For example, For example, the enhancer may include a CMV immediate-early enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the duplicated copies of the enhancer are separated by one or more sequences. In yet another embodiment, the expression cassette further includes an intron, such as a chicken beta-actin intron. Other suitable introns include those known in the art, such as those described in WO2011 / 126808. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize the mRNA. One example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence [see, e.g., MA Zanta-Boussif, et al., Gene Therapy (2009) 16:605-619].

[0073] These rAAVs are particularly suitable for gene delivery for therapeutic purposes and immunization, including inducing protective immunity. Furthermore, the compositions of the present invention can be used to produce desired gene products in vitro. For in vitro production, the desired product (e.g., protein) can be obtained from the desired culture after transfecting host cells with rAAV containing molecules encoding the desired product and culturing the cell culture under conditions that allow expression. The expressed product can then be purified and isolated, if desired. Suitable techniques for transfection, cell culture, purification, and isolation are known to those skilled in the art.

[0074] Therapeutic transgenes Useful products encoded by transgenes include various gene products that replace missing or defective genes, inactivate or "knock out," or "knock down" or reduce the expression of genes that are expressed at undesirably high levels, or deliver gene products with a desired therapeutic effect. In most embodiments, the treatment is "somatic cell gene therapy," i.e., the introduction of genes into cells of the body that do not produce sperm or eggs. In certain embodiments, the transgene-expressed protein has the sequence of a native human sequence. However, in other embodiments, synthetic proteins are expressed. Such proteins may be intended for human treatment, or in other embodiments, may be designed for the treatment of animals, including companion animals such as dog or cat populations, or for the treatment of livestock or other animals that come into contact with the human population.

[0075] Examples of suitable gene products may include those related to familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases.Examples of such rare diseases include spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (for example, methyl-CpG binding protein 2 (MeCP2), UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia (for example, frataxin), progranulin (PRGN) (related to non-Alzheimer's brain degeneration, including frontal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia).See, for example, www.orpha.net / consor / cgi-bin / Disease_Search_List.php;rarediseases.info.nih.gov / diseases.

[0076] Examples of suitable genes include, for example, insulin, glucagon, glucagon-like peptide-1 (GLP1), growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO) (including, for example, human, canine, or feline EPO), connective tissue growth factor (CTGF), neutrophil factors, such as basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), and the like. GF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), any one of the transforming growth factor alpha superfamily including TGFα, activin, inhibin, or any of the bone morphogenetic proteins (BMP) BMP1-15, the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, any one of agrin, semaphorin / collapsin, netrin 1 and netrin 2, hepatocyte growth factor (HGF), any one of the families of ephrin, noggin, sonic hedgehog, tyrosine hydroxylase.

[0077] Other useful transgene products include proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL), IL-1 through IL-36 (e.g., human interleukins IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12, IL-11, IL-12, IL-13, IL-18, IL-31, IL-35), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2 / flt3 ligand, etc. Gene products produced by the immune system are also useful in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. For example, in certain embodiments, rAAV antibodies can be designed to deliver canine or feline antibodies, such as anti-IgE, anti-IL31, anti-CD20, anti-NGF, and anti-GnRH. Useful gene products also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor protein (MCP), decay-accelerating factor (DAF), CR1, CF2, CD59, and C1 esterase inhibitor (C1-INH).

[0078] Still other useful gene products include any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. The present invention encompasses receptors for cholesterol control and / or lipid regulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very-low-density lipoprotein (VLDL) receptors, and scavenger receptors. The present invention also encompasses gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. Additionally, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD, and myogenin, ETS box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS binding proteins, STATs, GATA box binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

[0079] Other useful gene products include carbamoyl synthetase I, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase (ASL) for the treatment of argininosuccinate lyase deficiency, arginase, fumarate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, and aca These include rhesus alpha-fetoprotein (AFP), rhesus chorionic gonadotropin (CG), glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin gene products (e.g., mini- or micro-dystrophin). Still other useful gene products include enzymes that may be useful in enzyme replacement therapy, which is useful in various conditions caused by insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate may be used to treat lysosomal storage diseases (eg, suitable genes include those encoding β-glucuronidase (GUSB)).

[0080] In certain embodiments, rAAV can be used in gene editing systems, which can involve the co-administration of one rAAAV or multiple rAAV strains. For example, rAAV can be engineered to deliver SpCas9, SaCas9, ARCUS, Cpf1, and other suitable gene editing constructs.

[0081] Still other useful gene products include those used to treat hemophilia, including hemophilia B (including factor IX) and hemophilia A (including factor VIII and its variants, e.g., heterodimers and B-deleted domain light and heavy chains; U.S. Patent Nos. 6,200,560 and 6,221,349). In some embodiments, the minigene contains the first 57 base pairs of the factor VII heavy chain encoding a 10-amino acid signal sequence and the human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the minigene further contains the A1 and A2 domains, and 5 amino acids from the N-terminus of the B domain and / or the C-terminal 85 amino acids of the B domain, as well as the A3, C1, and C2 domains. In yet other embodiments, nucleic acids encoding the factor VIII heavy and light chains are provided in a single minigene separated by 42 nucleic acids encoding the 14 amino acids of the B domain [U.S. Patent No. 6,200,560].

[0082] Other useful gene products include non-natural polypeptides, such as chimeric or hybrid polypeptides with non-natural amino acid sequences containing insertions, deletions, or amino acid substitutions. For example, single-chain engineered immunoglobulins may be useful in certain immunodeficient patients. Other types of non-natural gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce overexpression of targets.

[0083] Reducing and / or modulating gene expression is particularly desirable for treating hyperproliferative conditions characterized by hyperproliferative cells, such as cancer and psoriasis. Target polypeptides include polypeptides produced exclusively or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, and fyn, as well as translocation genes bcr / abl, ras, src, p53, neu, trk, and EGRF. In addition to oncogene products as target antigens, target polypeptides for anti-cancer therapeutic and protective regimens include the variable regions of antibodies produced by B-cell lymphomas and the variable regions of T-cell receptors in T-cell lymphomas, and in some embodiments are also used as target antigens for autoimmune diseases. Other tumor-associated polypeptides can be used as target polypeptides, such as polypeptides found at higher levels in tumor cells, including polypeptides recognized by monoclonal antibody 17-1A and folate-binding polypeptides.

[0084] Other suitable therapeutic polypeptides and proteins include those that may be useful for treating individuals suffering from autoimmune diseases and disorders by conferring a broad protective immune response against targets associated with autoimmunity, including cellular receptors and cells that produce "self"-directed antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis. Each of these diseases is characterized by a T cell receptor (TCR) that binds to endogenous antigens and initiates the inflammatory cascade associated with autoimmune disease.

[0085] Additional exemplary genes that can be delivered via rAAV include, but are not limited to, glucose-6-phosphatase associated with glycogen storage disease or type 1A deficiency (GSD1), phosphoenolpyruvate-carboxykinase (PEPCK) associated with PEPCK deficiency, cyclin-dependent kinase-like 5 (CDKL5), also known as serine / threonine kinase 9 (STK9), associated with seizures and severe neurodevelopmental disorders, galactose-1-phosphate uridyltransferase associated with galactosemia, phenylalanine hydroxylase associated with phenylketonuria (PKU), branched-chain alpha-ketoacid dehydrogenase associated with maple syrup urine disease, fumarylacetoacetate hydrolase associated with tyrosinemia type 1, methylmalonyl-CoA mutase associated with methylmalonic acidemia, medium-chain acetyl-CoA associated with medium-chain acetyl-CoA deficiency, and methylmalonyl-CoA mutase associated with medium-chain acetyl-CoA deficiency. Cholesterol-CoA dehydrogenase, ornithine transcarbamylase (OTC) associated with ornithine transcarbamylase deficiency, argininosuccinate synthetase (ASS1) associated with citrullinemia, lecithin-cholesterol acyltransferase (LCAT) deficiency, methylmalonic acidemia (MMA), Niemann-Pick disease (type C1), propionic acidemia (PA), low-density lipoprotein receptor (LDLR) protein associated with familial hypercholesterolemia (FH), UDP-glucourosyltransferase associated with Crigler-Najjar disease, adenosine deaminase associated with severe combined immunodeficiency, hypoxanthine guanine phosphoribosyltransferase associated with gout and Leschnan syndrome, biotimidase associated with biotimidase deficiency, alpha-galactosidase A (α-Gal) associated with Fabry disease.A)), ATP7B associated with Wilson's disease, beta-glucocerebrosidase associated with Gaucher disease types 2 and 3, peroxisomal membrane protein 70 kDa associated with Zellweger syndrome, acetylsulfatase A (ARSA) associated with metachromatic leukodystrophy, galactocerebrosidase (GALC) enzyme associated with Krabbe disease, alpha-glucosidase (GAA) associated with Pompe disease, sphingomyelinase (SMPD1) gene associated with Niemann-Pick disease type A, argininosuccinate synthase associated with adult-onset type II citrullinemia (CTLN2), carbamoyl phosphate synthase 1 (CPS1) associated with urea cycle disorders, survival motor neuron (SMN) protein associated with spinal muscular atrophy, ceramidase associated with Farber lipogranulomatosis, b-hexosaminidase associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff disease, and Aspartyl-glucosaminidase associated with spartyl-glucosaminuria, a-fucosidase associated with fucosidosis, α-mannosidase associated with α-mannosidosis, porphobilinogen deaminase associated with acute intermittent porphyria (AIP), alpha-1 antitrypsin for the treatment of alpha-1 antitrypsin deficiency (emphysema), erythropoietin for the treatment of anemia due to thalassemia or renal failure, vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic disease, thrombomodulin and tissue factor pathway inhibitor for the treatment of blocked blood vessels, such as those found in atherosclerosis, thrombosis, or embolism, aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease, beta-adrenergic receptors for the treatment of congestive heart failure, phosphatase inhibitors, and phosphodiesterase inhibitors. These include antisense to holamban or its variants, sarcomeric (endoplasmic reticulum) adenosine triphosphatase-2 (SERCA2), and cardiac adenylate cyclase, tumor suppressor genes such as p53 for the treatment of various cancers, cytokines such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancer, dystrophin or mini-dystrophin and utrophin or mini-trophin for the treatment of muscular dystrophies, and insulin or GLP-1 for the treatment of diabetes.

[0086] In certain embodiments, the rAAVs described herein can be used in the treatment of mucopolysaccharidosis (MPS) disorders. Such rAAVs can include nucleic acid sequences encoding α-L-iduronidase (IDUA) for treating MPS I (Hurler, Hurler-Scheie, and Scheie syndromes), iduronate-2-sulfatase (IDS) for treating MPS II (Hunter syndrome), sulfamidase (SGSH) for treating MPS III A, B, C, and D (Sanfilippo syndrome), N-acetylgalactosamine-6-sulfate sulfatase (GALNS) for treating MPS IV A and B (Morquio syndrome), and N-acetylgalactosamine-6-sulfate sulfatase (GALNS) for treating MPS III A and B (Morquio syndrome). The nucleic acid sequence may include carrying a nucleic acid sequence encoding arylsulfatase B (ARSB) for treating MPS VI (Maroteaux-Lamy syndrome), a nucleic acid sequence encoding hyaluronidase for treating MPS I IX (hyaluronidase deficiency), and a nucleic acid sequence encoding beta-glucuronidase for treating MPS VII (Sly syndrome).

[0087] Immunogenic transgene In some embodiments, an rAAV vector containing a nucleic acid encoding a gene product associated with cancer (e.g., a tumor suppressor) can be used to treat cancer by administering the rAAV containing the rAAV vector to a subject with cancer. In some embodiments, an rAAV vector containing a nucleic acid encoding a small interfering nucleic acid (e.g., shRNA, miRNA) that inhibits the expression of a gene product associated with cancer (e.g., an oncogene) can be used to treat cancer by administering the rAAV containing the rAAV vector to a subject with cancer. In some embodiments, an rAAV vector containing a nucleic acid encoding a gene product associated with cancer (or a functional RNA that inhibits the expression of a gene associated with cancer) can be used for research purposes, for example, to study cancer or to identify therapeutic agents for treating cancer.The following is a non-limiting list of exemplary genes (e.g., oncogenes and tumor suppressors) known to be associated with the development of cancer: AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4 ,ATM,ATP5B,ATP5O,AXL,BARD1,BAX,BCL2,BHLHB2,BLMH,BRAF,BRCA1,BRCA2,BTK,CANX,CAP1,CAPN1,CAPNS1,CAV1,C BFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CI B1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1 , CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1, DVL3, E2F1, E2F3, E2F5 , EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FK. BP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2, GNB2L1, GPR39, GRB 2、GSK3A、GSPT1、GTF2I、HDAC1、HDGF、HMMR、HPRT1、HRB、HSPA4、HSPA5、HSPA 8、HSPB1、HSPH1、HYAL1、HYOU1、ICAM1、ID1、ID2、IDUA、IER3、IFITM1、IGF1R、 IGF2R、IGFBP3、IGFBP4、IGFBP5、IL1B、ILK、ING1、IRF3、ITGA3、ITGA6、ITGB 4、JAK1、JARID1A、JUN、JUNB、JUND、K-ALPHA-1、KIT、KITLG、KLK10、KPNA2、KR AS2、KRT18、KRT2A、KRT9、LAMB1、LAMP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF 、LYN、LZTR1、MADH1、MAP2K2、MAP3K8、MAPK12、MAPK13、MAPKAPK3、MAPRE1、MA RS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3, MYB, MYBL1, MYBL2, MYC, MYCL1, M YCN、MYD88、MYL9、MYLK、NEO1、NF1、NF2、NFKB1、NFKB2、NFSF7、NID、NINE、NM BR、NME1、NME2、NME3、NOTCH1、NOTCH2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR 2F6、NRAS、NRG1、NSEP1、OSM、PA2G4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PD GFA、PDGFB、PDGFRA、PDPK1、PEA15、PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3C B、PIK3CG、PIM1、PKM2、PKMYT1、PLK2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A 、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP、PRSS15、PSMA1、PTCH、PTEN、PTGS1、PTMA, PTN, PTPRN, RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RARA, RARB, RASGRF1, RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6 KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, sphingomyelin phosphodiesterase 1 (SMPD1), SNAI2, SND1, SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, T K1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1, TOB1, TP53, TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101 , TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9. ,

[0088] The rAAV vector may contain, as a transgene, a nucleic acid encoding a protein or functional RNA that regulates apoptosis. The following are genes related to apoptosis, as well as genes encoding the products of these genes and their homologs, and genes that inhibit the expression of these genes and their homologs, which are useful as transgenes in certain embodiments of the invention: Results from a range of shRNAs and miRNAs:R PS27A、ABL1、AKT1、APAF1、BAD、BAG1、BAG3、BAG4、BAK1、BA X, BCL10, BCL2, BCL2A1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC 2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD 14 NOD2, NOD1, CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CAS P9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, ​​MCL1, NOL3 PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNF RSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, and TNFSF1 8, CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5.

[0089] Useful transgene products also include miRNAs. miRNAs and other small interfering nucleic acids regulate gene expression through cleavage / degradation of target RNA transcripts or translational repression of target messenger RNAs (mRNAs). miRNAs are typically naturally expressed as final 19-25 untranslated RNA products. miRNAs exert their activity through sequence-specific interactions with the 3' untranslated region (UTR) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors, which are then processed into miRNA duplexes and further into "mature" single-stranded miRNA molecules. This mature miRNA guides the multiprotein complex miRISC, which identifies target sites on target mRNAs, for example, within the 3' UTR region, based on complementarity with the mature miRNA.

[0090] The following non-limiting list of miRNA genes and their homologs are useful as targets for transgenes or small interfering nucleic acids encoded by transgenes (e.g., miRNA sponges, antisense oligonucleotides, TuD RNAs) in certain embodiments of the following methods: hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*, hsa-let-7f, hsa-let-7g, hsa-let-7h*, hsa-let-7i, hsa-let-7j, hsa-let-7j, hsa-let-7j, hsa-let-7i ... -7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*, hsa-let-71, hsa-let-71*, hsa-miR-1, hsa-mi R-100, hsa-miR-100*, hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hs a-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107, hsa-miR-10a, hsa-miR -10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178, hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-m iR-1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-120 7-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122, hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p, hsa-miR-1225-5p, hs a-miR-1226, hsa-miR-1226*, hsa-miR-1227, hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233, hsa-miR-1234, hsa-miR- 1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124, hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246, hsa-miR-1247, hsa- miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b, hsa-miR-12 56、hsa-miR-1257、hsa-miR-1258、hsa-miR-1259、hsa-miR-125a-3p、hsa- miR-125a-5p、hsa-miR-125b、hsa-miR-125b-1*、hsa-miR-125b-2*、hsa-mi R-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262, hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267, hs a-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272, hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b, hsa-mi R-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278, hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287, hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR- 1291, hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298 hsa-miR-1299 hsa-miR-1300 hsa-miR-1301 hsa-miR-1302 hsa-miR-1303 hsa-miR-1304 hsa-miR-1305 hsa-miR-1306 hsa-miR-1307 hsa-miR -1308、hsa-miR-130a、hsa-miR-130a*、hsa-miR-130b、hsa-miR-130b*、hsa-miR-132、hsa-miR-132*、hsa-miR-1321、hsa-miR-1322、hsa-miR-1323、h sa-miR-1324, hsa-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b, hsa-miR-135b*, hsa-miR-136, hsa-miR-1 36*, hsa-miR-137, hsa-miR-138, hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR -141, hsa-miR-141*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hs a-miR-146a, hsa-miR-146a*, hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b, hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa,-miR-148b*、hsa-miR-149、hsa-miR-149*、hsa-miR-150、hsa-miR-150*、hsa-miR-151-3p、hsa-miR-151-5p、hsa-miR-152、hsa-miR-153、hsa-miR-1 54, hsa-miR-154*, hsa-miR-155, hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*, hsa-miR-16, hsa-miR-16-1*, hsa-miR- 16-2*、hsa-miR-17、hsa-miR-17*、hsa-miR-181a、hsa-miR-181a*、hsa-miR-181a-2*、hsa-miR-181b、hsa-miR-181c、hsa-miR-181c*、hsa-miR-181d hsa-miR-182 hsa-miR-182* hsa-miR-1825 hsa-miR-1826 hsa-miR-1827 hsa-miR-183 hsa-miR-183* hsa-miR-184 hsa-miR-185 hsa-miR-18 5*, hsa-miR-186, hsa-miR-186*, hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a, hsa-miR-18a*, hsa-miR-18b, hsa- miR-18b*, hsa-miR-190, hsa-miR-190b, hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR- 193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*, hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b, hsa-miR-197, hsa -miR-198、hsa-miR-199a-3p、hsa-miR-199a-5p、hsa-miR-199b-5p、hsa-miR-19a、hsa-miR-19a*、hsa-miR-19b、hsa-miR-19b-1*、hsa-miR-19b-2*、hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b, hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*, hsa-miR-203, hsa-mi R-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a, hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*, hsa-miR-21, hsa-miR- 21*, hsa-miR-210, hsa-miR-211, hsa-miR-212, hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR-2 18, hsa-miR-218-1*, hsa-miR-218-2*, hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22, hsa-miR-22*, hsa-miR-220a, hsa-miR -220b, hsa-miR-220c, hsa-miR-221, hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*, hsa-miR-224, hsa-miR-23a, hsa-mi R-23a*, hsa-miR-23b, hsa-miR-23b*, hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*, hsa-miR-26a, hsa-miR-26a-1*, hsa- miR-26a-2*, hsa-miR-26b, hsa-miR-26b*, hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p, hsa-miR-28-5p, hsa-miR-296 -3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298, hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b, hsa-miR-296-1*hsa-miR-296 -2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300, hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b, hsa-miR-302b*, hsa -miR-302c、hsa-miR-302c*、hsa-miR-302d、hsa-miR-302d*、hsa-miR-302e、hsa-miR-302f、hsa-miR-30a、hsa-miR-30a*、hsa-miR-30b、hsa-miR-30 b*、hsa-miR-30c、hsa-miR-30c-1*、hsa-miR-30c-2*、hsa-miR-30d、hsa-m iR-30d*、hsa-miR-30e、hsa-miR-30e*、hsa-miR-31、hsa-miR-31*、hsa-mi R-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p, hsa-miR-32 4-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329, hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p, hsa-miR-335 hsa-miR-335* hsa-miR-337-3p hsa-miR-337-5p hsa-miR-338-3p hsa-miR-338-5p hsa-miR-339-3p hsa-miR-339-5p hsa-miR-33a hsa-miR- 33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340, hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346, hsa-miR-34a, hsa -miR-34a*、hsa-miR-34b、hsa-miR-34b*、hsa-miR-34c-3p、hsa-miR-34c-5p、hsa-miR-361-3p、hsa-miR-361-5p、hsa-miR-362-3p、hsa-miR-362-5p、hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371-3p, hsa-m iR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*, hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375, hsa-miR-376a hsa-miR-376a* hsa-miR-376b hsa-miR-376c hsa-miR-377 hsa-miR-377* hsa-miR-378 hsa-miR-378* hsa-miR-379 hsa-miR-379* hsa-miR-3 80, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411, hsa-miR -411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, h sa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*, hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a, h sa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p, hsa-m iR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p, hsa-miR-487ahsa-miR-4、 87b, hsa-miR-488, hsa-miR-488*, hsa-miR-489, hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p, hsa-miR-492, hsa-miR-49 3, hsa-miR-493*, hsa-miR-494, hsa-miR-495, hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p, hsa-miR-499-5p, hsa-m iR-500, hsa-miR-500*, hsa-miR-501-3p, hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503, hsa-miR-504, hsa-miR-505, hsa-miR -505*, hsa-miR-506, hsa-miR-507, hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p, hsa-miR-509-5p, hsa-miR-510, hsa-m iR-511, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b, hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p, hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*, hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a -3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c*、h sa-miR-518d-3p、hsa-miR-518d-5p、hsa-miR-518e、hsa-miR-518e*、hsa- miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p, hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*, hsa-miR-520a-3phsa-miR-520a-5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR-520d-3p、h sa-miR-520d-5p、hsa-miR-520e、hsa-miR-520f、hsa-miR-520g、hsa-miR- 520h, hsa-miR-521, hsa-miR-522, hsa-miR-523, hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p, hsa-miR-526b, hsa-miR-52 6b*、hsa-miR-532-3p、hsa-miR-532-5p、hsa-miR-539、hsa-miR-541、hsa-miR-541*、hsa-miR-542-3p、hsa-miR-542-5p、hsa-miR-543、hsa-miR-544 hsa-miR-545 hsa-miR-545* hsa-miR-548a-3p hsa-miR-548a-5p hsa-miR-548b-3p hsa-miR-5486-5p hsa-miR-548c-3p hsa-miR-548c-5p hs a-miR-548d-3p、hsa-miR-548d-5p、hsa-miR-548e、hsa-miR-548f、hsa-mi R-548g、hsa-miR-548h、hsa-miR-548i、hsa-miR-548j、hsa-miR-548k、hsa -miR-5481、hsa-miR-548m、hsa-miR-548n、hsa-miR-548o、hsa-miR-548p、hsa-miR-549、hsa-miR-550、hsa-miR-550*、hsa-miR-551a、hsa-miR-551b hsa-miR-551b* hsa-miR-552 hsa-miR-553 hsa-miR-554 hsa-miR-555 hsa-miR-556-3p hsa-miR-556-5p hsa-miR-557 hsa-miR-558 hsa-miR -559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570, hsa-miR-571hsa-miR-572、hsa-miR-5、 73, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578, hsa-miR-579, hsa-miR-580, h sa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587, hsa-miR-588, hsa-miR-589 hsa-miR-589* hsa-miR-590-3p hsa-miR-590-5p hsa-miR-591 hsa-miR-592 hsa-miR-593 hsa-miR-593* hsa-miR-595 hsa-miR-596 hsa-miR -597, hsa-miR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606, hsa-miR-607 hsa-miR-608 hsa-miR-609 hsa-miR-610 hsa-miR-611 hsa-miR-612 hsa-miR-613 hsa-miR-614 hsa-miR-615-3p hsa-miR-615-5p hsa-miR-6 16, hsa-miR-616*, hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624, hsa-miR-624* hsa-miR-625 hsa-miR-625* hsa-miR-626 hsa-miR-627 hsa-miR-628-3p hsa-miR-628-5p hsa-miR-629 hsa-miR-629* hsa-miR-630 hsa-miR -631, hsa-miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-m iR-652, hsa-miR-653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa- miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668, hsa-miR-671-3p, hsa-miR-671-5p, h sa-miR-675, hsa-miR-7, hsa-miR-708, hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744, hsa-miR-744*, hsa-miR-758, hsa- miR-760, hsa-miR-765, hsa-miR-766, hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p, hsa-miR-769-3p, hsa-miR-769-5p, hsa -miR-770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p, hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-m iR-877*, hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR- 890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9, hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923hsa-miR, -924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, h sa-miR-93*, hsa-miR-933, hsa-miR-934, hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, h miR-939, hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944, hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a, hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*. For example, miRNAs targeting chromosome 8 open reading frame 72 (C9orf72), which expresses superoxide dismutase (SOD1), which is associated with amyotrophic lateral sclerosis (ALS), are of interest.

[0091] MiRNA inhibits the function of the target mRNA, thereby inhibiting the expression of the polypeptide encoded by the mRNA. Therefore, blocking the activity of miRNA (partially or completely) (e.g., silencing the miRNA) can effectively induce or restore the expression of the inhibited polypeptide (de-repressing the polypeptide). In one embodiment, de-repression of the polypeptide encoded by the mRNA target of miRNA is achieved by inhibiting miRNA activity in cells through any one of a variety of methods. For example, blocking the activity of miRNA can be achieved by hybridizing with a small interfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge, TuD RNA) that is complementary or substantially complementary to the miRNA, thereby blocking the interaction of the miRNA with its target mRNA. As used herein, a small interfering nucleic acid that is substantially complementary to a miRNA is a nucleic acid that can hybridize with the miRNA and block the activity of the miRNA. In some embodiments, the small interfering nucleic acid that is substantially complementary to miRNA is a small interfering nucleic acid that is complementary to miRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases. "miRNA inhibitor" is a drug that blocks the function, expression, and / or processing of miRNA. For example, these molecules include, but are not limited to, microRNA-specific antisense, microRNA sponge, tough decoy RNA (TuD RNA), and microRNA oligonucleotides (double-stranded, hairpin, short oligonucleotide) that inhibit miRNA interaction with Drosha complex.

[0092] Still other useful transgenes may include those encoding immunoglobulins that confer passive immunity to pathogens. An "immunoglobulin molecule" is a protein containing the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain, which are covalently linked to each other and capable of specifically combining with an antigen. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The terms "antibody" and "immunoglobulin" may be used interchangeably herein.

[0093] An "immunoglobulin heavy chain" is a polypeptide that contains at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of the variable region of an immunoglobulin heavy chain or at least a portion of the constant region of an immunoglobulin heavy chain. Thus, an immunoglobulin-derived heavy chain has significant regions of amino acid sequence homology with members of the immunoglobulin gene superfamily. For example, the heavy chain in a Fab fragment is an immunoglobulin-derived heavy chain.

[0094] An "immunoglobulin light chain" is a polypeptide that comprises at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of the variable region or at least a portion of the constant region of an immunoglobulin light chain. Thus, an immunoglobulin-derived light chain is It has significant regions of amino acid homology with members of the gene superfamily.

[0095] "Immunoadhesins" are chimeric, antibody-like molecules that combine the functional domain of a binding protein, usually a receptor, ligand, or cell adhesion molecule, with immunoglobulin constant domains, usually including the hinge and Fc regions.

[0096] A "fragment antigen-binding" (Fab) fragment is the region of an antibody that binds to an antigen. It consists of the constant and variable domains of each of the heavy and light chains.

[0097] The anti-pathogen constructs are selected based on the causative agent (pathogen) of the disease against which protection is sought. These pathogens may be viral, bacterial, or fungal in origin and may be used to prevent infection in humans for human diseases or in non-human mammals or other animals for veterinary diseases.

[0098] The rAAV may contain genes encoding antibodies, particularly neutralizing antibodies against viral pathogens. Such anti-viral antibodies may include anti-influenza antibodies directed against one or more of influenza A, influenza B, and influenza C. Type A viruses are the most virulent human pathogens. Influenza A serotypes associated with pandemics include H1N1, which caused the Spanish flu in 1918, H2N2, which caused the swine flu in 2009, H2N2, which caused the Asian flu in 1957, H3N2, which caused the Hong Kong flu in 1968, and H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7, which caused the avian flu in 2004. Other target pathogenic viruses include arenaviruses (including Funin, Machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picornaviridae (including rhinoviruses and echoviruses), coronaviruses, paramyxoviruses, morbilliviruses, respiratory syncytial viruses, togaviruses, coxsackieviruses, JC virus, parvovirus B19, parainfluenza, adenoviruses, reoviruses, varicella virus (variola major (smallpox)), and vaccinia virus (cowpox) and varicella-zoster (pseudorabies) from the poxvirus family. Viral hemorrhagic fevers are caused by members of the arenavirus family (Lassa fever) (a family also related to lymphocytic choriomeningitis (LCM)), filoviruses (Ebola virus), and hantaviruses (Premara). Members of the picornavirus family (the Rhinoviridae subfamily) are associated with the common cold in humans. The coronavirus family includes multiple non-human viruses, such as infectious bronchitis virus (poultry), porcine transmissible gastrointestinal virus (pigs), porcine hemagglutinin encephalomyelitis virus (pigs), feline infectious peritonitis virus (cats), feline enteric coronavirus (cats), and canine coronavirus (dogs). Human respiratory coronaviruses are presumed to be related to the common cold, non-A, B, or C hepatitis, and sudden acute respiratory syndrome (SARS).The paramyxovirus family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubulavirus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, morbilliviruses including measles and canine distemper, and pneumonia viruses including respiratory syncytial virus (RSV). The parvovirus family includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The adenovirus family includes viruses that cause respiratory diseases (e.g., AD7, ARD, OB). Thus, in certain embodiments, the rAAV vectors described herein can be engineered to express anti-Ebola antibodies, e.g., 2G4, 4G7, 13C6, anti-influenza antibodies, e.g., FI6, CR8033, and anti-RSV antibodies, e.g., palivizumab, motavizumab.

[0099] Neutralizing antibody constructs against bacterial pathogens may also be selected for use in the present invention. In one embodiment, the neutralizing antibody construct is directed against the bacteria itself. In another embodiment, the neutralizing antibody construct is directed against a toxin produced by the bacteria. Examples of airborne bacterial pathogens include, for example, Neisseria meningitidis (meningitis), Klebsiella pneumoniae (pneumonia), Pseudomonas aeruginosa (pneumonia), Burkholderia pseudomallei (pneumonia), Burkholderia mallei (pneumonia), Acinetobacter (pneumonia), Staphylococcus catarrhalis, Moraxella lacunar, Alcaligenes, Cardiobacterium, Haemophilus influenzae (influenza), Haemophilus parainfluenzae, Bordetella pertussis (whooping cough), Francisella tularensis (pneumonia, fever), Legionella pneumoniae (Legionella pneumoniae), and the like. disease), Chlamydia psittacosis (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis (tuberculosis (TB)), Mycobacterium kansasii (TB), Mycobacterium avium (pneumonia), Nocardia asteroides (pneumonia), Bacillus anthracis (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacterium diphtheriae (diphtheria), and Mycoplasma pneumoniae (pneumonia).

[0100] rAAV can contain genes encoding antibodies, particularly neutralizing antibodies against bacterial pathogens such as the causative agent of anthrax, the toxin produced by Bacillus anthracis. Neutralizing antibodies against protectant (PA), one of the three peptides that form the toxoid, have been described. The other two polypeptides consist of lethal factor (LF) and edema factor (EF). Anti-PA neutralizing antibodies have been described as effective for passive immunization against Bacillus anthracis. See, for example, U.S. Patent No. 7,442,373; R. Sawada-Hirai et al., J Immune Based Ther Vaccines. 2004;2:5. (online 2004 May 12). Still other anti-anthrax toxin neutralizing antibodies have been described and / or can be generated. Similarly, neutralizing antibodies against other bacteria and / or bacterial toxins can be used to generate AAV-delivered anti-pathogen constructs as described herein.

[0101] Antibodies against infections may be raised by parasites or fungi, including, for example, Aspergillus, Absidiacolymbifera, Rhizopus, Mucor pulveus, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Penicillium species, Micropolyspora phenii, Thermoactinomyces vulgaris, Alternaria alternata, Cladosporium species, Helminthosporium, and Stachybotrys species.

[0102] rAAV may contain genes encoding antibodies, particularly neutralizing antibodies, against pathogenic factors of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), GBA-associated Parkinson's disease (GBA-PD), rheumatoid arthritis (RA), irritable bowel syndrome (IBS), chronic obstructive pulmonary disease (COPD), cancer, tumors, systemic sclerosis, asthma, and other diseases. Such antibodies include, but are not limited to, alpha-synuclein, anti-vascular endothelial growth factor (VEGF) (anti-VEGF), anti-VEGFA, anti-PD-1, anti-PDL1, anti-CTLA-4, anti-TNF-alpha, anti-IL-17, anti-IL-23, anti-IL-21, anti-IL-6, anti-IL-6 receptor, anti-IL-5, anti-IL-7, anti-Factor XIII, anti-IL-2, anti-HIV, anti-IgE, anti-tumor necrosis factor receptor-1 (TNFR1), anti-Notch 2 / 3, anti-Notch 1, anti-OX40, anti-erb-b2 receptor tyrosine kinase 3 (ErbB3), anti-ErbB2, anti-beta cell growth factor receptor 1 (BETA-1), ...17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti-IL-17, anti- Antibodies may be selected from the group consisting of: mature antigen, anti-B lymphocyte stimulating factor, anti-CD20, anti-HER2, anti-granulocyte macrophage colony-stimulating factor, anti-oncostatin M (OSM), anti-lymphocyte activation gene 3 (LAG3) protein, anti-CCL20, anti-serum amyloid P component (SAP), anti-prolyl hydroxylase inhibitor, anti-CD38, anti-glycoprotein IIb / IIIa, anti-CD52, anti-CD30, anti-IL-1 beta, anti-epidermal growth factor receptor, anti-CD25, anti-RANK ligand, anti-complement system protein C5, anti-CD11a, anti-CD3 receptor, anti-alpha-4 (α4) integrin, anti-RSV F protein, and anti-integrin α4β7. Still other pathogens and diseases will be apparent to those skilled in the art. Other suitable antibodies include, for example, among others, Antibodies useful for treating Alzheimer's disease may include anti-beta-amyloid (e.g., crenezumab, solanezumab, adacunamab), anti-beta-amyloid fibrils, anti-beta-amyloid plaques, anti-tau, bapineuzamab, etc. Other suitable antibodies for treating various indications include, for example, those described in PCT / US2016 / 058968, filed October 27, 2016, published as WO2017 / 075119A1.

[0103] rAAV vector production For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the expression cassette can be carried on any suitable vector, e.g., a plasmid, that is delivered to a packaging host cell. Plasmids useful in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic, insect, or mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art.

[0104] Methods for generating and isolating AAV suitable for use as a vector are known in the art. Generally, see, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10:717-733, and the references cited below, each of which is incorporated herein by reference in its entirety. To package a transgene into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette. The cap and rep genes can be supplied in trans.

[0105] In one embodiment, the expression cassettes described herein are engineered into genetic elements (e.g., shuttle plasmids) that introduce the immunoglobulin construct sequences carried thereon into packaging host cells to produce viral vectors. In one embodiment, the selected genetic elements can be delivered to AAV packaging cells by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be generated. Alternatively, the expression cassettes can be used to generate viral vectors other than AAV or for the in vitro production of antibody mixtures. Methods used to generate such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0106] The term "AAV9 intermediate" or "AAV9 vector intermediate" refers to an assembled rAAV capsid that lacks the desired genomic sequence packaged therein. These may be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of the gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0107] The recombinant adeno-associated virus (AAV) described herein can be produced using known techniques. For example, see WO2003 / 042397; WO2005 / 033321; WO2006 / 110689; US7588772 B2. Such methods include culturing host cells containing an expression cassette consisting of a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, at least an AAV inverted terminal repeat (ITR) and a transgene, and sufficient helper functions to allow the expression cassette to be packaged into AAV capsid proteins. Methods for producing capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. For example, see Gao, et al., Proc. Natl. Acad. Sci. USA 100(10), 6081-6086(2003) and US2013 / 0045186A1.

[0108] In one embodiment, a producer cell culture useful for producing recombinant AAV is provided. Such cell culture comprises a nucleic acid that expresses AAV capsid proteins in a host cell, a nucleic acid molecule suitable for packaging into an AAV capsid, such as a vector genome containing AAV ITRs, and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence that directs the expression of the product in the host cell, as well as sufficient AAV rep function and adenovirus helper function to enable packaging of the nucleic acid molecule into a recombinant AAV capsid. In one embodiment, the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., baculovirus).

[0109] Optionally, the rep function is provided by an AAV other than the AAV providing the capsid, for example, but not limited to, AAV1 rep protein, AAV2 rep protein, AAV3 rep protein, AAV4 rep protein, AAV5 rep protein, AAV6 rep protein, AAV7 rep protein, AAV8 rep protein, or rep 78, rep 68, rep 52, rep The AAV capsid sequences may be rep40, rep68 / 78, and rep40 / 52, or fragments thereof. Optionally, the rep and cap sequences are located on the same genetic element in cell culture. A spacer may be present between the rep sequence and the cap gene. Any of these AAV or mutant AAV capsid sequences may be under the control of exogenous regulatory sequences that direct their expression in host cells.

[0110] In one embodiment, the cells are produced in suitable cell culture (e.g., HEK293) cells. Methods for producing the gene therapy vectors described herein include methods well known in the art, such as producing the plasmid DNA used to produce the gene therapy vector, producing the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the produced plasmids are an AAV cis-plasmid encoding the AAV genome and the gene of interest, an AAV trans-plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process can include method steps such as initiating cell culture, passaging the cells, seeding the cells, transfecting the cells with plasmid DNA, changing the medium to serum-free medium after transfection, and recovering the vector-containing cells and culture medium. The recovered vector-containing cells and culture medium are referred to herein as crude cell harvest. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of which are incorporated herein by reference in their entireties: 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065.

[0111] The crude cell harvest is then subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector.

[0112] A two-step high salt affinity chromatography purification, followed by anion exchange resin chromatography, is used to purify the vector drug product and remove empty capsids. These methods are described in detail in International Patent Application No. PCT / US2016 / 065970, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,071, filed April 13, 2016, and U.S. Patent Application No. 62 / 226,357, filed December 11, 2015, entitled "Scalable Purification Method for AAV9," which are incorporated herein by reference. Purification methods for AAV8 are described in International Patent Application No. PCT / US2016 / 065976, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,098, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,341, filed December 11, 2015, and for rh10, in International Patent Application No. PCT / US16 / 66013, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,055, filed April 13, 2016, and U.S. Patent Application No. 62 / 322,055, filed December 11, 2015, and "Scalable Purification Method for AAV8." No. 62 / 266,347, entitled "Scalable Purification Method for AAVrhlO," and for AAV1, see International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,083, filed April 13, 2016, and U.S. Patent Application No. 62 / 26,351, filed December 11, 2015, entitled "Scalable Purification Method for AAVl," which are incorporated herein by reference in their entireties.

[0113] To calculate empty and full particle content, the VP3 band volume for a selected sample (e.g., in the examples herein, an iodixanol gradient-purified preparation, where the number of GC = the number of particles) is plotted against the number of GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test article peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the particle to genome copy ratio (pt / GC). Pt / mL - GC / mL gives empty pt / mL. Empty pt / mL is divided by pt / mL, then multiplied by 100 to obtain the percentage of empty particles.

[0114] Generally, assay methods for AAV vector particles with empty capsids and packaged genomes are well known in the art.See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128.To test for denatured capsids, the method can be, for example, denatured capsids in 3-8% Tris- This method involves subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel containing acetate, and then running the gel until the sample material is separated and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as the primary antibody to bind to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and includes a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to the antibody, most preferably a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semi-quantitatively measure the binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method capable of detecting radioisotope radiation, electromagnetic radiation, or color change, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, samples from the column fractions are collected and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid proteins are resolved on a precast gradient polyacrylamide gel (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, such as SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another appropriate nuclease) to remove exogenous DNA. After inactivation of the nucleases, the sample is further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for the DNA sequence between the primers.The number of cycles required to reach a predetermined level of fluorescence (threshold cycle, Ct) was measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector was used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample was used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0115] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serum protease, such as Protease K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with Protease K buffer and treated with Protease K, followed by heat inactivation. Suitably, the sample is diluted with a volume of Protease K buffer equal to the sample size. The Protease K buffer may be concentrated two-fold or more. Typically, the Protease K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally carried out at about 55°C for about 15 minutes, but may be carried out at lower temperatures (e.g., about 37°C to about 50°C) for longer periods (e.g., about 20 to about 30 minutes) or at higher temperatures (e.g., up to about 60°C) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally carried out at about 95°C for about 15 minutes, but may be carried out at lower temperatures (e.g., about 70 to about 90°C) and for longer periods (e.g., about 20 to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0116] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for measuring single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, for example, M. Lock et al., Hu et al. See Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0117] Briefly, a method for separating rAAV particles containing packaged genome sequences from genome-defective AAV intermediates involves subjecting a suspension containing recombinant AAV viral particles and AAV capsid intermediates to high-performance liquid chromatography, in which the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at a high pH and subjected to a salt gradient while monitoring the eluate for ultraviolet absorbance at about 260 and about 280. The pH can be adjusted depending on the AAV selected. See, for example, WO2017 / 160360 (AAV9), WO2017 / 100704 (AAVrhlO), WO2017 / 100676 (e.g., AAV8), and WO2017 / 100674 (AAVl), which are incorporated herein by reference. In this method, AAV complete capsids are recovered from the fraction eluting when the A260 / A280 ratio reaches the infection point. In one example, for an affinity chromatography step, the diafiltered product may be applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures the AAV2 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flows through the column, while the AAV particles are efficiently captured.

[0118] Compositions and Uses Provided herein are compositions comprising at least one rAAV stock (e.g., an rAAV stock or a mutant rAAV stock) and optional carriers, excipients, and / or preservatives. An rAAV stock refers to multiple rAAV vectors that are the same, for example, in the amounts described below in the discussion of concentrations and dosage units.

[0119] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present invention into suitable host cells. In particular, rAAV vector-delivered transgenes can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0120] In one embodiment, the composition comprises the final formulation suitable for delivery to subject, for example, an aqueous liquid suspension that is buffered to physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to subject.In other embodiments, the composition can be lyophilized and reconstituted when administered.

[0121] A suitable surfactant, or combination of surfactants, can be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl-terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), can be used. Copolymers such as poloxamer, SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycapric acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol may be selected. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by a three-digit number, where the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0122] The vector is administered in an amount sufficient to transfect cells and provide a sufficient level of gene transfer and expression to provide a therapeutic effect without undue adverse effects or with a medically acceptable physiological effect, which can be determined by those skilled in the art. Conventional and pharmaceutically acceptable administration routes include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parent administration routes. Administration routes may be combined if desired.

[0123] The dosage of a viral vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore may vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1 x 10, which is about 25 to about 1000 microliters to about 100 mL. 9 ~1×10 16 The range of the solution containing the concentration of genome viral vector.Dosage can be adjusted to balance therapeutic benefit against any side effects, and this dosage can vary depending on the therapeutic use that recombinant vector is used for.The expression level of transgene can be monitored to determine the dosage frequency that results in viral vector, preferably the AAV vector that contains minigene.Optionally, the dosage regimen that is similar to that described for therapeutic purposes can be used for immunization using the composition of the present invention.

[0124] The replication-defective virus composition is formulated in a dosage unit, and for a human patient, it is about 1.0 x 10 9 GC~approx. 1.0×10 16 GC, including amounts of replication-defective virus (to treat an average subject weighing 70 kg) in the range of 1.0×10, and all integers or fractions within that range, preferably 1.0×10 12 GC~1.0×10 14 In one embodiment, the composition contains at least 1 x 10 per dose, including all integers or fractions within the range. 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x1010 , 8x10 10 , or 9x10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , or 9x10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , or 9x10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 1 4 , 6x10 14 , 7x10 14 , 8x10 14 , or 9x10 14In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or fractions within the range. 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15 In one embodiment, for human applications, the dose is 1 x 10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It can be in the range of GC.

[0125] These above-mentioned doses may be administered in various volumes of the carrier, excipient, or buffer formulation, ranging from about 25 to about 1000 microliters, or higher volumes including all numbers within that range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 375 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 550 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 650 μL. In another embodiment, the volume is about 700 μL. In another embodiment, the volume is about 700-1000 μL.

[0126] In certain embodiments, the dose is about 1 x 10 9 GC / g brain mass ~ approx. 1x10 12 GC / g brain mass. In certain embodiments, the dose is about 3x10 10 GC / g brain mass ~ approx. 3x10 11 GC / g brain mass. In certain embodiments, the dose is about 5x10 10 GC / g brain mass ~ approx. 1.85x10 11 GC / g brain mass.

[0127] In one embodiment, the viral construct comprises at least about 1 x 10 9 GC~approx. 1×10 15 , or approximately 1 × 10 11 ~5×10 13 The GC may be delivered in doses of 1000 mg / kg / day. Appropriate volumes and concentrations for delivery of these doses can be determined by one of skill in the art. For example, a volume of about 1 μL to 150 mL may be selected, although higher volumes may be selected for adults. Typically, for newborn infants, an appropriate volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, a volume of up to about 30 mL may be selected. For preteens and teens, a volume of up to about 50 mL may be selected. In yet other embodiments, patients may receive intrathecal administration in a selected volume of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL. Other appropriate volumes and dosages may be determined. Dosage may be adjusted to balance therapeutic benefit against any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is being utilized.

[0128] The recombinant vectors described above can be delivered to host cells according to published methods. The rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient. In certain embodiments, for administration to a human patient, the rAAV is administered in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. The formulation is preferably suspended in a solution containing cerebrospinal fluid (CSF). Preferably, the formulation is adjusted to a physiologically acceptable pH, for example, pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Because the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH within this range may be desirable for intrathecal delivery, and a pH of about 6.8 to about 7.2 may be desirable for intravenous delivery. However, other pH values ​​within a broader range, and subranges of these, may be selected for other delivery routes.

[0129] In another embodiment, the composition includes a carrier, diluent, excipient, and / or adjuvant. A suitable carrier can be readily selected by one skilled in the art, taking into account the indication for which the transduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should contain components that prevent rAAV from adhering to the infusion tubing but do not interfere with rAAV binding activity in vivo. A suitable surfactant or combination of surfactants can be selected from non-toxic, non-ionic surfactants. In one embodiment, a primary hydroxyl-terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400. Other surfactants and poloxamers, i.e., nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycapric acid glyceride), polyoxy-oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol, may be selected. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by three digits, where the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.In one example, the formulation can contain a buffered saline solution, e.g., containing one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate 7H2O), potassium chloride, calcium chloride (e.g., calcium chloride 2H2O), dibasic sodium phosphate, and mixtures thereof, in water. Preferably, for intrathecal delivery, the osmolality is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, commercially available diluents can be used as suspending agents or in combination with other suspending agents and any other excipients. See, e.g., Elliotts B® Solution [Lukare Medical]. In other embodiments, the formulation can contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0130] Optionally, in addition to the rAAV and carrier, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0131] The composition according to the present invention may comprise a pharmaceutically acceptable carrier as defined above. Preferably, the composition described herein comprises one or more AAVs suspended in an effective amount of a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed for delivery to a subject via injection, osmotic pump, intrathecal catheter, or by another device or route. In one embodiment, the composition is formulated for intrathecal delivery.

[0132] As used herein, the terms "intrathecal delivery" or "intrathecal administration" refer to the route of administration of a drug by injection into the spinal canal, more particularly, by injection into the subarachnoid space to reach the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital / intracisternal, and / or C1-2 puncture. For example, material may be introduced via lumbar puncture for diffusion throughout the subarachnoid space. In another example, injection may be intracisternal.

[0133] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the route of administration of a drug directly into the cisternal cerebellar-medullary cerebrospinal fluid, more particularly, by a permanently placed tube via suboccipital puncture or by direct injection into the cisterna magna.

[0134] In one embodiment, the vectors provided herein can be administered intrathecally via a method and / or device. See, for example, WO2017 / 181113, which is incorporated herein by reference. Alternatively, other devices and methods can be selected. The method includes advancing a spinal needle into a patient's cisterna magna, connecting a length of flexible tubing to the proximal hub of the spinal needle and connecting the outlet port of a valve to the proximal end of the flexible tubing, and after the advancing and connecting steps, allowing the tubing to self-prime with the patient's cerebrospinal fluid, then connecting a first container containing a quantity of isotonic solution to the flush inlet port of the valve, and then connecting a second container containing a quantity of a pharmaceutical composition to the vector inlet port of the valve. After connecting the first and second containers to the valve, a passage for fluid flow is opened between the vector inlet port and the outlet port of the valve, and the pharmaceutical composition is injected into the patient through the spinal needle; after injection of the pharmaceutical composition, a passage for fluid flow is opened through the flush inlet port and the outlet port of the valve, and an isotonic solution is injected into the spinal needle to flush the pharmaceutical composition into the patient.

[0135] This method and this device, respectively, can optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices can be used for such intrathecal delivery.

[0136] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0137] The terms "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist," "consisting," and variations thereof are to be interpreted exclusively rather than inclusively. Although various embodiments herein are set forth using the word "comprising," in other circumstances, the relevant embodiment may be referred to using the words "consisting of" or "consisting essentially of." It is also intended to be interpreted and described using the leaves.

[0138] As used herein, the term "about" means a variability of 10% (±10%) from a given reference, unless otherwise specified.

[0139] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

[0140] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published documents which provide general guidance to those skilled in the art for many of the terms used herein.

[0141] The term "expression" is used herein in the broadest sense and includes the production of RNA or RNA and protein. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or proteins. Expression can be transient or stable.

[0142] As used herein, the term "NAb titer" refers to the production of neutralizing antibodies (e.g., anti-AAV Nabs) that neutralize the physiological effects of the targeted epitope (e.g., AAV). Anti-AAV NAb titers can be measured, for example, as described in Calcedo, R., et al., "Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses." Journal of Infectious Diseases, 2009. 199(3): pp. 381-390, which is incorporated herein by reference.

[0143] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a coding sequence, a promoter, and optionally other regulatory sequences therefor, which can be delivered to a packaging host cell by a genetic element (e.g., a plasmid) and packaged into a viral vector capsid (e.g., a viral particle). Typically, such expression cassettes for producing viral vectors contain coding sequences for gene products described herein adjacent to packaging signals of the viral genome, and other expression control sequences, such as those described herein.

[0144] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. During infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," (August 2001), Vol. 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0145] As used herein, the term "operably linked" refers to an expression control sequence that is contiguous with a gene of interest and acts in trans on the gene of interest or controls the expression of the gene of interest. It refers both to expression control sequences that act at a distance to control genes in a gene.

[0146] The term "heterologous," when used with reference to a protein or nucleic acid, indicates that the protein or nucleic acid contains two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids having two or more sequences from unrelated genes arranged to create a new functional nucleic acid are typically produced recombinantly. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene. Thus, with respect to the coding sequence, the promoter is heterologous.

[0147] A "replication-deficient virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged into a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are replication-deficient, i.e., they are unable to produce progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy because replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0148] In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endo- and exo-nucleases can be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded and / or double-stranded DNA, and RNA. Such steps can involve a single nuclease or a mixture of nucleases directed against different targets, which can be endonucleases or exonucleases.

[0149] The term "nuclease-resistant" indicates that the AAV capsid is constructed entirely around an expression cassette designed to deliver a transgene into a host cell and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present from the production process.

[0150] The term "translation" in the context of the present invention relates to the process at the ribosome, whereby an mRNA chain controls the assembly of an amino acid sequence to produce a protein or peptide.

[0151] As used throughout this specification and claims, the terms "comprising" and "including" are inclusive of other components, elements, integers, steps, etc. Conversely, the term "consisting" and variations thereof exclude other components, elements, integers, steps, etc.

[0152] As stated above, the term "about," when used to modify a numerical value, means a variance of ±10%, unless otherwise specified.

[0153] The following examples are illustrative only and are not intended to limit the invention. [Example]

[0154] The following examples report extensive deamidation in AAV8 and seven additional diverse AAV serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on vector age and multiple primary sequence and 3D structural factors, but was largely independent of vector recovery and purification conditions. We demonstrate the potential for deamidation to affect vector transduction activity and correlate early loss of vector activity at several AAV8 asparagines with rapidly progressing spontaneous deamidation. We explore mutational strategies to stabilize side chain amides, improve vector transduction, and reduce lot-to-lot molecular variability, a key concern in biologics manufacturing. This study demonstrates a previously unknown aspect of AAV capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

[0155] Example 1 below provides characterization of post-translational modifications to the AAV8 vector capsid by one- and two-dimensional gel electrophoresis, mass spectrometry, and de novo structural modeling. Following identification of multiple putative deamidation sites on the capsid surface, the impact on capsid structure and function is assessed both in vitro and in vivo. Example 1 further extends this analysis to AAV9 to determine whether this phenomenon applies to serotypes other than AAV8, confirming that AAV capsid deamidation is not serotype-specific. Examples 2 and 3 exemplify deamidation in additional AAVs.

[0156] Example 4 relates to novel epitopes mapped on the AAV9 capsid.

[0157] Example 1: Deamidation of amino acids on the surface of adeno-associated virus capsids A. Materials and Methods 1. 1D and 2D gel electrophoresis For 1D SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, AAV vectors were first incubated for 80 min in the presence of lithium dodecyl sulfate and a reducing agent. o The gel was denatured at 3°C ​​for 20 minutes. It was then run on a 4-12% Bis-Tris gel at 200V for 90 minutes and stained with Coomassie blue. For the data in Figures 1A-1D, 2D gel electrophoresis was performed by Kendrick Laboratories, Inc. (Madison, WI). For subsequent experiments, 2D SDS-PAGE was performed in-house. For this purpose, 3 x 10 nucleotides were diluted in 150 μL of phosphate-buffered saline (PBS) containing 35 mM NaCl and 1 mM MgCl2. 11 The GC AAV vector and 500U Turbo Nuclease marker (Accelagen, San Diego, CA) were combined and used for 37 o The samples were then incubated at -80°C for 10 minutes. Nine volumes of absolute ethanol were then added, the samples were vortexed, and the samples were stored at -80°C for 10 minutes. o Incubate at least 2 hours at 4°C, then incubate on ice for 5 minutes at maximum speed for 15 minutes. oThe supernatant was decanted, the pellet air-dried, and then resuspended in Resuspension Buffer #1 [0.15% SDS, 50 mM dithiothreitol (DTT), 10 mM Tris pH 7.5, and 1 μL pH 6-9 ampholyte, ThermoFisher ZM0023, in ddH2O] and incubated gently at room temperature. After 30 minutes, the sample tube was flicked to mix, 1 μg of chicken conalbumin marker (Sigma Aldrich, St. Louis, MO) was added, and the sample was incubated at 37°C. o The samples were incubated at 4°C for 30 minutes and flicked to mix at 15 minutes. o Transfer to 95°C for 15-20 min, vortex, and o The samples were incubated at 4°C for 2.5 minutes, cooled, then centrifuged at maximum speed for 1 minute and briefly vortexed. Next, 10 μL of each sample was mixed with 140 μL of Resuspension Buffer #2 (9.7 M urea, 2% CHAPS, 0.002% bromophenol blue, and 0.05% ampholytes as described above in ddH2O) and incubated at room temperature for 10 minutes. The mixtures were then applied to pH 6-10 immobilized pH gradient (IPG) strips (ThermoFisher, Waltham, MA) and run on a ZOOM IPGRunner system according to the manufacturer's instructions. The following isoelectric focusing parameters were used: 0.1 W per strip The electrophoresis was performed at 100-1,000 V for 120 minutes, 1,000-2,000 V for 120 minutes, and 2,000 V for 120 minutes, with a current limit of 0.05 mA. The IPG strips were then reduced and loaded into single-well 4-12% Bis-Tris gels and run in 1D as described above. The relative migration of AAV VP was determined relative to the internal control proteins turbonuclease (Acelagen, 27 kDa) and chicken egg white conalbumin (Sigma-Aldrich, 76 kDa, pI 6.0-6.6).

[0158] 2. Vector Production The University of Pennsylvania Vector Core generated recombinant AAV vectors for 1D and 2D gel electrophoresis and mass spectrometry experiments and purified them on cesium chloride or iodixanol gradients as previously described. (Lock M, et al. Hum Gene Ther 2010;21(10):1259-71; Gao GP, et al. Proc Natl Acad Sci USA. 2002;99(18):11854-9. Affinity-purified vector was produced as follows: HEK293 cells were grown in ten 36-layer HyperStack vessels (Corning) and cotransfected with a mixture of the vector genome plasmid (pAAV-LSP-IVS2.hFIXco-WPRE-bGH), a trans plasmid containing the AAV2 rep and AAV8 cap genes, and an adenovirus helper plasmid. PEIpro (PolyPlus) was used as the transfection reagent. Five days after transfection, the supernatant was collected, clarified through a Sartoguard PES Midicap filter (Sartorious Stedim), treated with benzonase (Millipore), and then salt was added to 0.6M. The clarified bulk harvest material was concentrated 10-fold by tangential flow filtration (TFF) and then diafiltered against four volumes of affinity column loading buffer. The vector was captured on a POROS Capture Select (ThermoFisher) affinity column, and the vector peak was eluted directly into neutralization buffer at low pH. The neutralized eluate was diluted into high-pH binding buffer and loaded onto an anion-exchange polishing column (Cimultus QA-8; Bia Sprestions) to enrich the preparation for genome-containing (intact) particles. Intact vector particles were eluted with a shallow salt elution gradient and immediately neutralized. Finally, the vector was subjected to a second TFF for final concentration and buffer exchange into formulation buffer (PBS + 0.001% Pluronic F-68).

[0159] Mutant vectors for in vitro assays were produced by small-scale triple transfection of HEK293 cells in 6-well plates. Plasmid DNA (0.091 μg cis plasmid, 0.91 μg trans plasmid, and 1.82 μg deltaF6 Ad helper plasmid in 90 μL of serum-free medium) was mixed with 5.6 μL of 1 mg / mL polyethyleneimine solution and 90 μL of serum-free medium, incubated at room temperature for 15 minutes, and added to the cells. 0.8 mL of fresh serum-free medium was added. The following day, 0.5 mL of the upper medium was replaced with full-serum medium. Three days after transfection, the vector was recovered by three freeze / thaw cycles, followed by centrifugation to remove cell debris and supernatant. The cis plasmid contains a transgene cassette encoding a firefly luciferase transgene under the control of a chicken beta-actin (CB7) promoter with a promega chimeric intron and a rabbit beta globin (RBG) polyadenylation signal. The trans-plasmid encoded the wtAAV8 cap gene, and the Quikchange Lightning Mutagenesis Kit (Agilent Technologies, Wilmington, DE) was used to generate mutant AAV8 cap variants. The vectors were titered as previously described (Lock M, et al. Hum Gene Ther 2010;21(10):1259-71).

[0160] For time-course vector generation experiments, vectors were generated by medium-scale triple transfection of HEK293 cells in 15 cm tissue culture dishes. 36 μL of 1 μg / mL polyethyleneimine solution in 1 mL of serum-free medium was mixed with plasmid DNA (0.6 μg cis plasmid, 5.8 μg trans plasmid, 11.6 μg DeltaF6 Ad-helper plasmid), incubated at room temperature for 15 minutes, and added to cells at approximately 60% confluency on plates refreshed with 14 mL of serum-free medium. The next day, 8 mL of top medium was replaced with fresh, complete serum medium. The vector was recovered by collecting all the top medium, scraping the cells from the dish, and freezing them at -80°C. Crude vector was recovered from the supernatant / cell mixture by applying three freeze / thaw cycles and clarifying the lysate by centrifugation. The vector was purified and concentrated for mass spectrometry analysis by adding benzonase, 1 M Tris pH 7.5, and 5 M NaCl to the clarified lysate to a final concentration of 20 mM Tris and 360 mM NaCl. The vector was captured on a 1 ml POROS CaptureSelect affinity column, and the vector peak was eluted directly into neutralization buffer at low pH. Fractions were analyzed by absorbance at 280 nm, and the most concentrated fractions were subjected to mass spectrometry.

[0161] For in vivo experiments, vectors were produced using the previously described wtAAV8 capsid or one of six deamidation mutants, and the transgene cassette contains the CB7 promoter, PI intron, firefly luciferase transgene, and RBG polyadenylation signal (Lock M, et al. Hum Gene Ther 2010;21(10):1259-71).

[0162] 3. Perform Mass Spectrometry / Digest / Analysis Materials: Ammonium bicarbonate, DTT, iodoacetamide (IAM), and 18O-enriched water (97.1% purity) were purchased from Sigma (St. Louis, MO), and acetonitrile, formic acid, trifluoroacetic acid (TFA), 8 M guanidine hydrochloride (GndHCl), and trypsin were purchased from Thermo Fischer Scientific (Rockford, IL).

[0163] Trypsin digestion: A stock solution of 1 M DTT and 1.0 M iodoacetamide was prepared. Capsid proteins were denatured and incubated for 90 min in the presence of 10 mM DTT and 2 M GndHCl. o The denatured protein solution was reduced at 37°C for 10 minutes. The sample was allowed to cool to room temperature and then alkylated using 30 mM IAM at room temperature for 30 minutes in the dark. 1 mL DTT was added to quench the alkylation reaction. 20 mM ammonium bicarbonate (pH 7.5-8) was added to the denatured protein solution in an amount that would dilute the final GndHCl concentration to 200 mM. Trypsin solution was added to achieve a trypsin-to-protein ratio of 1:20, and the denatured protein solution was then cooled to 37°C for 10 minutes. o The mixture was incubated overnight at C. After digestion, TFA was added to a final concentration of 0.5% to quench the digestion reaction.

[0164] For the 18O-water experiments, capsid samples were first buffer-exchanged into 100 mM ammonium bicarbonate prepared in 18O-water using a Zeba spin desalting column (Thermo Scientific, Rockford, IL). Two buffer exchanges were performed to completely remove water from the samples. Stock solutions of 1 M DTT and 1 M IAM were prepared in 18O-water. The same denaturation, alkylation, and digestion steps described above were followed using the 18O-water reagents and buffers.

[0165] Liquid chromatography tandem mass spectrometry: Online chromatography was performed using a Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific) coupled with a Q Exactive HF equipped with an Acclaim PepMap column (15 cm long, 300 μm internal diameter) and a NanoFlex source (Thermo Fisher Scientific). The column temperature was maintained at 35°C during online analysis. Mobile phase A (containing 0.1% formic acid) was used. Peptides were separated with a gradient of mobile phase B (acetonitrile with MilliQ water) and mobile phase B (acetonitrile with 0.1% formic acid). The gradient was run from 4% B to 6% B over 15 minutes, to 10% B in 25 minutes (40 minutes total), and then to 30% B in 46 minutes (86 minutes total). Samples were loaded directly onto the column. The column dimensions were 75 cm x 15 μm ID and packed with 2 micron C18 media (Aclame PepMap). Due to the loading, loading, and wash steps, the total time for each liquid chromatography-tandem mass spectrometry run was approximately 2 hours.

[0166] Mass spectrometry data were acquired using a data-dependent top-20 method on a Q Exactive HF mass analyzer to dynamically select the most abundant unsequenced precursor ions from a survey scan (m / z 200–2000). Sequencing was performed via higher energy collisional dissociation fragmentation with a target value of 1e5 ions determined by predictive automatic gain control, and precursor isolation was performed in a 4 m / z window. Survey scans were acquired at m / z 200 with a resolution of 120,000. The HCD spectral resolution was set to 30,000 at m / z 200 with a maximum ion injection time of 50 ms and a normalized collision energy of 30. The RF level of the S lens was set to 50, which optimally transmits the m / z region occupied by peptides from the digest. Precursor ions that were single, unassigned, or had charge states greater than six were excluded from fragmentation selection.

[0167] Data processing: BioPharma Finder 1.0 software (Thermo All acquired data were analyzed using a Fischer Scientific (Fischer Scientific). For peptide mapping, searches were performed using a single-entry protein FASTA database with carbamidomethylation as the fixed modification and oxidation, deamidation, and phosphorylation as variable modifications. Tandem mass spectrometry spectra were analyzed with a mass accuracy of 10 ppm, high protease specificity, and a confidence level of 0.8. Because deamidation adds 0.984 Da (the mass difference between the -OH and -NH groups) to the mass of the intact molecule, mass spectrometric identification of deamidated peptides is relatively straightforward. The deamidation rate for a given peptide was determined by dividing the mass area of ​​the deamidated peptide by the sum of the areas of the deamidated and native peptides. Considering the number of possible deamidation sites, isotonic species deamidated at different sites may comigrate in a single peak. Therefore, fragment ions derived from peptides with multiple potential deamidation sites can be used to identify or distinguish between multiple deamidation sites. In these cases, the relative intensities within the observed isotope patterns can be used to specifically determine the relative abundance of different deamidated peptide isomers. This method assumes that the fragmentation efficiency for all isomeric species is the same and independent at the site of deamidation. This approach allows for the definition of the specific sites involved in deamidation and the potential combinations involved in deamidation.

[0168] Secondary data processing: Secondary analysis of raw mass spectra was performed at the University of Maryland, Baltimore County, using the following methods. Peaks Studio v5.3 software (Bioinformatics Solutions Inc.) was used for all mass analyses. Data refinement of raw data files was performed using parameters of a precursor m / z tolerance of ≤10 ppm, a minimum of 2, and a maximum of 4 precursor charge states. De novo sequencing of input spectra was performed using the Peaks algorithm with a precursor ion error tolerance of 10 ppm and a product ion error tolerance of 0.1 Da. The digestion enzyme was set as trypsin, variable modifications were oxidation, phosphorylation, and deamidation, and the fixed modification was carbamidomethylation of cysteine.

[0169] 4. Structural analysis of AAV capsid AAV8 atomic coordinates, structure factors, and A structure refinement was performed to generate electron density independent of the primary amino acid sequence of AAV8 VP3 for use in three-dimensional (3D) structural analysis of the capsid. This analysis was performed to observe electron density for the isoaspartic acids of the AAV8 capsid that was not biased by the predicted primary sequence of AAV8 VP3. Using the resulting structure, the four asparagines in the AAV8 VP3 primary sequence were modeled, with the N+1 glycine as the isoaspartic acid. The AAV8 capsid structure was then refined using Crystallography and NMR Systems (CNS) software by rigorously forcing an icosahedral amorphous matrix using a standard refinement protocol (Brunger AT, et al. Acta Crystallogr D Biol Crystallogr 1998;54(Pt 5):905-21). A structural model of the isoaspartic acid was obtained from the HIC-UP database, followed by generating a molecular dictionary in PRODRG for structural refinement (Kleywegt GJ Acta Crystallogr D Biol Crystallogr 2007;63(Pt 1):94-100). An average electron density map of the AAV8 capsid (including the CNS) was then calculated and visualized using COOT software, after which minor adjustments were made to the resulting model to fit the modeled isoaspartic acid residue into the electron density map (Emsley P and Cowtan K Acta Crystallogr D Biol Crystallogr 2004;60(Pt 12 Pt 1):2126-32). This protocol was repeated to further model N512 in the AAV9 VP3 primary sequence, which contains the N + 1 glycine (PDB number: 3UX1). All figures were generated using COOT, PyMol, and UCSF Chimera (Emsley P and Cowtan K Acta Crystallogr D Biol Crystallogr 2004;60(Pt 12 Pt 1):2126-32, DeLano WL PyMOL: An Open-Source Molecular Graphics Tool Vol.40,2002:82-92, Pettersen EF,et al.J Comput Chem 2004;25(13):1605-12). To compare the electron density map of deamidated isoaspartic acid residues with the modeled isoaspartic acid residues of AAV8 and AAV9, we obtained several structures of previously identified deamidated proteins (PDB numbers: 1DY5, 4E7G, 1RTU, 1W9V, 4E7D, and 1C9D) (Rao FV, et al. Chem Biol 2005;12(1):65-76; Noguchi S, et al. Biochemistry 1995;34(47):15583-91; Esposito L, et al. J Mol Biol 2000;297(3):713-32).

[0170] The temperature coefficients of deamidated residues were determined by averaging the temperature coefficients of each atom of each asparagine residue reported in the atomic coordinates of the AAV8 or AAV9 crystal structure (PDB numbers: 3RA8, 3UX1).

[0171] 5.Animal research The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal procedures. To evaluate vector performance, 8-week-old C57BL / 6 mice were intravenously injected with 3 e10 GC of wtAAV8 or capsid-mutated vectors in a volume of 100 μL via tail vein injection. All mice were sacrificed on day 14. For in vivo evaluation of luciferase expression, mice (approximately 20 g) were anesthetized and intraperitoneally injected with 200 μL or 15 mg / mL luciferin substrate (Perkin Elmer, Waltham, MA). Mice were imaged 5 minutes after luciferin administration via an IVIS Xenogen in vivo imaging system. Signal in the region of interest was quantified using Living Image 3.0 software. Measurements were taken on days 7 and 14.

[0172] 6. Assessment of Mutant Vector Titer and In Vitro Transduction Efficiency Vector titers were determined by qPCR of DNAse I-resistant genomes. qPCR primers anneal to the polyadenylation sequences of the packaged transgene. For in vitro evaluation of vector transduction efficiency by luciferase expression, 0.9e5 Huh7 cells / well were seeded in black-walled 96-well plates in complete DMEM (10% fetal bovine serum, 1% penicillin / streptomycin). The following day, the medium was removed and replaced with 50 μL of crude or purified vector diluted in complete medium. For each crude vector sample, four dilutions were tested in a 3-fold dilution series. After 48 h, 0.3 μg / μL of luciferin (Promega, Madison, WI) in complete medium was prepared and added to the transduced cells in a volume of 50 μL. Results were read using a Biotek Clarity luminometer. We found that luciferase activity / GC added to target cells was constant over a wide range of GC but saturable at high MOIs. Therefore, the linearity of the dilution series data (luminescence units vs. GC) was examined, the highest point where saturation was evident was excluded, and the average luciferase / GC was calculated for values ​​within the linear range of each assay for each variant. This yielded a transduction efficiency value. Data were normalized to simplify comparison by setting the Wt control to a value of 1.

[0173] 7. Distribution in the body DNA was extracted from liver samples using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany), and then vector GC DNA was analyzed by real-time PCR with a primer / probe set designed against the RBG polyadenylation signal of the transgene cassette as previously described (Chen SJ, et al. Hum Gene Ther Clin Dev 2013;24(4):154-60). [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0174] B. Results AAV8 exhibits substantial charge heterogeneity in its capsid proteins. To qualitatively assess the presence of post-translational modifications on AAV8 vector capsids that may affect vector performance, we analyzed AAV8 total capsid proteins purified by iodixanol gradients by both 1D and 2D gel electrophoresis. In 1D reduced sodium dodecyl sulfate (SDS) gels, VP1, VP2, and VP3 resolved as single bands at the appropriate molecular weights (Figure 1B) (Rose JA, et al. J Virol 1971;8(5):766-70). Further evaluation by 2D gel electrophoresis, which separates proteins based on charge (Figure 1C), revealed that each capsid protein was further resolved as a series of distinct spots with different isoelectric points (pI) ranging from pH 6.3 to >7.0, depending on the VP isoform (Figure 1D). Individual spots for each VP were separated by discrete intervals of 0.1 pI units when measured as migration relative to an internal isoelectric point standard for carbonic anhydrase isoforms, suggesting a single residue charge change. The existence of these isoforms suggests that each VP has the potential to undergo multiple modifications, thereby resulting in different migrations under isoelectric focusing.

[0175] Deamidation, in which a portion of the (typically asparagine) side chain amide groups are converted to carboxylic acids (Figure 1A), is a common source of charge isomerism in protein preparations. To determine whether deamidation is responsible for the distinct populations of VP charge isoforms, two AAV8 asparagine residues were individually mutated to aspartate. These capsid mutations require a charge shift equivalent to the complete deamidation of a single additional asparagine residue. 2D gel analysis of the mutants shows that the major spots for VP1, VP2, and VP3 shifted one spot position more acidic (0.1 pH units) than the equivalent spots in wild-type (wt) AAV8 (Figure 1E-G). The magnitude of this shift corresponds to the observed spacing between wt VP charge isoforms. Therefore, the 2D gel pattern of the AAV capsid protein is consistent with multi-site deamidation.

[0176] Spontaneous deamidation occurs in AAV8 vector capsids Identifying the modifications responsible for the distinct spotting patterns for each capsid protein To identify the deamidation pattern, we analyzed a panel of AAV8 vectors by mass spectrometry. Coverage of the AAV8 capsid protein averaged over 95% of the total VP1 sequence (data not shown). We detected extensive deamidation of a subset of asparagine and glutamine residues by mass spectrometry, indicating an approximately 1 Da increase in the observed mass of individual peptides compared to the predicted value based on the DNA-encoded sequence. We observed this deamidation pattern in all preparations of AAV8 vectors (Figure 2A-D).

[0177] To assess the overall heterogeneity of deamidation among commonly used purification methods and examine deamidation in the VP1 and VP2 specific regions, nine lots of AAV8 produced by triple transfection in 293 cells were selected and purified by either a cesium chloride gradient, an iodixanol gradient, or affinity chromatography. The vectors also varied in terms of promoter and transgene cassette. To determine whether the presence of the vector genome affected deamidation, AAV8 preps produced by triple transfection in 293 cells in the absence of the cis-plasmid (producing only empty capsids) were also evaluated and purified by an iodixanol gradient.

[0178] There was widespread deamidation across asparagine and glutamine residues in the AAV8 capsid, ranging from undetectable to over 99% of individual amino acids being deamidated (Figure 2E). The highest level of deamidation (over 75%) occurred at asparagine residues where the N+1 residue was glycine (i.e., N+1 vs. N+1) (Table 1). Lower levels of deamidation (i.e., up to 17%) were detected at additional asparagine residues where the N+1 residue was not glycine. Average deamidation at asparagine was fairly consistent across preparations. We also detected deamidation at glutamine residues, but at a lower frequency than asparagine, with the highest percentage observed being less than 2% at Q467 (Figure 7). This observation was inconsistent across preparations (data not shown). The greatest preparation-to-preparation difference was observed at residue N499 (where the N+1 residue is asparagine), with values ​​ranging from less than 1% to over 50% deamidation. In any case, the observed variation in deamidation between preparations of vector did not appear to be related to the purification method, transgene identity, or the presence of the vector genome, suggesting that these factors do not affect the deamidation rate. [Table 11]

[0179] Next, we performed a series of experiments to determine whether sample processing contributed to the observed levels of deamidation in AAV8. Extreme temperature (70°C for 7 days) or pH (pH 2 or pH 10 for 7 days) did not significantly induce additional deamidation in AAV8 capsids (Figure 4A and Figure 4B). Given this resistance, it is unlikely that the observed deamidation occurred solely during the purification step, which was brief and relatively mild. We attempted to perform mass spectrometry on unpurified vector to determine the extent of deamidation before and after purification, but were unsuccessful. Similarly, a deuterium oxide control indicates that processing inherent in the mass spectrometry workflow does not contribute to additional deamidation events (Figure 4C).

[0180] To validate our mass spectrometry workflow, we investigated two recombinant proteins that had previously been evaluated for deamidation, and the findings (Figure 5A and Figure 5B) are consistent with published results [Henderson, LE, Henriksson, D, and Nyman, PO (1976). Primary structure of human carbonic anhydrase C. The Journal of biological chemistry 251:5457-5463, and Carvalho, RN, Solstad, T, Bjorgo, E, Barroso, JF, and Flatmark, T (2003). Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of Asn-->Asp mutant forms.The Journal of biological Chemistry 278:15142-1515]. Additionally, we engaged a secondary laboratory to evaluate the raw data from AAV8. This independent analysis identified identical deamidated sites and confirmed the accuracy of the analysis, which was due to software-to-software variability in peak detection and area calculation. Variation in the degree of correction at each site was minimal (Figure 6).

[0181] Structural topography, temperature factors, and identity of the N+1 amino acid contribute to deamidation frequency The structure of AAV8 has been solved and published (PDB identifier: 2QA0) (Nam HJ, et al. J Virol 2011;85(22):11791-99). We next examined the AAV8 capsid structure for evidence of favorable conditions for non-enzymatic deamidation and correlated deamidation rates with established structural features (Nam HJ, et al. J Virol 2007;81(22):12260-71). We focused exclusively on asparagine residues because factors influencing asparagine deamidation are better characterized in the literature and asparagine deamidation events are much more common than glutamine deamidation events (Robinson, NE, and Robinson, AB (2001). Molecular clocks. Proc Natl Acad Sci USA 98:944-949). The temperature (or B) factors of each of these residues were also determined from the AAV8 crystal structure; the temperature factor is a measure of the displacement of an atom from its average position, with higher values ​​indicating larger displacements, higher thermal vibrations, and therefore increased flexibility (Parthasarathy S and Murphy MR. Protein Science: A Publication of the Protein Society 1997;6:2561-7). The majority of target asparagines are located within or near surface-exposed HVRs (Table 1), providing a structurally favorable, solvent-exposed environment for deamidation (Govindasamy L, et al. J Virol 2013;87(20):11187-99). We found that residues located in these flexible loop regions are more frequently deamidated on average than residues in less flexible regions, such as beta strands and alpha helices. For example, the N-G residue at position N263, part of HVR I, has a high temperature factor and is deamidated on average >98% of the time (Figure 7A and Figure 6, Table 1). N514, which deamidated approximately 85% of the time (Figures 3 and 6, Table 1), is also in an HVR with an N+1 glycine (HVR V); however, its local temperature factor is relatively low compared to N263 due to interactions with residues on other VP monomers in the 3-fold axis. Unfavorable +1 residues and lower local temperature factors correlate with lower deamidation, even for HVR residues. For example, N517 is only 4% deamidated on average (Table 1); this residue has a temperature factor comparable to that of highly deamidated N514, but its N+1 residue is a serine, reducing the likelihood of a deamidation event due to steric hindrance. This indicates that several factors cumulatively determine the degree of deamidation at a given capsid position, but the identity of the +1 residue is clearly the most influential factor.

[0182] To test the role of the +1 residue in asparagine deamidation, we generated mutant vectors in which the AAV8 NG site was individually mutated at the +1 position to either alanine or serine. Model peptide studies indicate that NG peptides deamidate with a half-life as short as one day, whereas NA or NS peptides typically deamidate 25- or 16-fold slower, respectively (Robinson NE and Robinson AB. Proc Natl Acad Sci USA. 2001;98(8):4367-72). Mass spectrometry analysis of vector mutants confirmed the central role of the +1 site in determining the extent of vector deamidation. NG sites in this set (greater than 80% deamidated in the wild-type) showed selective stabilization of the adjacent asparagine when the +1 site was changed to alanine (less than 5% deamidation) or serine (less than 14% deamidation) (Table 2). [Table 12]

[0183] Residues located in regions that were at least partially buried, not readily exposed to solvent, and / or had low local flexibility in the intact, fully assembled AAV8 capsid were less frequently deamidated than those located in more favorable environments (Table 1). Nevertheless, some residues in unfavorable states were deamidated. For example, N630, while at least partially buried, still had a detectable degree of deamidation. The presence of phenylalanine as the N+1 residue for this residue suggests that this region may be a novel site for nonenzymatic autoproteolytic cleavage in the AAV8 VP3 protein.

[0184] Structural modeling of AAv8 VP3 confirms the deamidation event To provide direct evidence of deamidation in the context of assembled capsids, the crystal structure of AAV8 was evaluated (Nam HJ, et al. J Virol 2011;85(22):11791-9). The resolution of the available crystal structure of this serotype (i.e., 2.7 Å) is not high enough to identify the terminal atoms in the R group and therefore is insufficient to directly distinguish between asparagine, aspartic acid, and isoaspartic acid residues. Other aspects of the structure of aspartic acid isomers formed under these conditions provided an opportunity to determine deamidation from the 2.7 Å structure. This analysis is based on two assumptions: 1) the primary product of spontaneous deamidation of asparagine is isoaspartic acid, not aspartic acid, and is produced in a 3:1 ratio (Geiger T and Clarke SJ Biol Chem 1987;262(2):785-94); 2) asparagine or aspartic acid can be distinguished from isoaspartic acid by the shorter length of the electron density map corresponding to the R group of isoaspartic acid. This shorter R group is created when the beta carbon from the R group of isoaspartic acid is lost during decomposition of the succinimidyl intermediate during the deamidation reaction as it is incorporated into the main chain of the AAV8 VP3 capsid protein backbone.

[0185] We first refined the AAV8 structure itself to generate AAV8 capsid electron density unbiased by the known AAV8 VP3 sequence. We then examined the purified AAV8 crystal structure for evidence of deamidation based on the presence of shorter R groups associated with isoaspartic acid (Figure 3A-E). The electron density map confirmed shorter R groups for the highly deamidated N+1 glycine residues at positions 263 (Figure 3C), 385 (not shown), 514 (Figure 3D), and 540 (Figure 3E), compared with the asparagine at position 410, which had no deamidation detected by mass spectrometry (Figure 3B). Therefore, the deamidation indicated by the electron density map is consistent with the data generated by mass spectrometry, which showed greater than 75% deamidation at these sites. The resulting isoaspartic acid model was comparable to isoaspartic acid residues observed in the crystal structures of other known deamidated proteins, confirming the validity of the AAV8 analysis (Rao FV, et al. Chem Biol. 2005; 12(1): 65-76, Noguchi S, et al. Biochemistry 1995; 34(47): 15583-91, Esposito L, (J Mol Biol 2000;297(3):713-32). This structural analysis serves as an independent confirmation of the deamidation phenomenon observed when analyzing AAV8 capsids via mass spectrometry.

[0186] AAV capsid deamidation is not serotype specific For evidence of capsid deamidation, we investigated serotypes beyond AAV8. We investigated AAV9 vector preparations using 2D gel electrophoresis (Figure 11A) and mass spectrometry (Figure 11B), including controls for potential vector treatment effects (Figure 11D-F). The pattern and extent of AAV9 deamidation was similar to that of AAV8. All four AAV9 NG sites were greater than 85% deamidated, while the 13 non-NG sites were deamidated to a lesser extent, with some sites showing high lot-to-lot variability in deamidation percentage. We then applied a structural analysis workflow to refit existing AAV9 crystal data (Figure 11C, Table 3). Similar to AAV8, isoaspartic acid residues fit better with the electron density of several NG sites within the AAV9 crystal structure. 2D gel analysis (data not shown) and mass spectrometry (summarized in Table 4) were further extended to five evolutionarily diverse serotypes (rh32.33, AAV7, AAV5, AAV4, AAV3B, and AAV1). All of the capsids examined contained similar patterns and degrees of deamidation, indicating that this modification is widespread in clinically relevant AAV vectors and is determined by similar underlying primary sequence and structural factors. [Table 13] [Table 14]

[0187] Deamidation events may affect capsid assembly and transduction efficiency One approach to testing the functional impact of deamidation is to replace asparagine with aspartate by genetic mutation. We generated aspartate-mutated vectors encoding luciferase reporters for each deamidated AAV8 asparagine by small-scale triple transfection of 293 cells, and titrated the vectors by qPCR of DNAse I-resistant genome copies (Figure 8A). The mutations had little effect on capsid recombination compared to wtAAV8, and the effect was limited to mostly buried non-NG sites, where overall deamidation in the wt vector was low. We then evaluated the mutation panel for in vitro transduction efficiency in human liver-derived Huh7 cells (Figure 8B). Several mutants showed reduced transduction efficiency, with positions N57, N94, N263, N305, Q467, N479, and N653 showing a greater than 10-fold loss of transduction. We observed a similar number of susceptible sites for AAV9 (Figure 11G and Figure 11H). Because typically only a fraction of residues at a given position are endogenously deamidated, this approach has the potential to overestimate loss of function in proteins such as capsids, whose functional units are homomeric assemblies; endogenous modification at one capsid site may be compensated for by adjacent subunits with intact residues. Nevertheless, we reasoned that this method could be useful for prioritizing deamidated residues for future monitoring during production or mutational stabilization. Functional data from a population of endogenously deamidated vectors would be needed to place this loss-of-function mutagenesis data in the appropriate context.

[0188] Loss of vector activity over time correlates with progressive deamidation Given the apparently short half-life of NG deamidation, we reasoned that vector samples differing in age by only 1 day would exhibit distinct deamidation profiles, providing an opportunity to correlate endogenous deamidation with function. Our large-scale vector preparation protocol requires triple transfection of 293 cells, followed by 5 days of incubation for vector production, and 1–2 days for vector purification. To approximate this process, we prepared medium-scale triple transfections of 293 cells (each 10 x 15 cm cell culture dish) with wt AAV8. Vectors (2 x 15 cm cell culture dishes / day) were collected at 1-day intervals for 5 days, and time points were maintained until the end of the 5-day period by freezing the vectors at -80 °C. We then assessed crude vector titers and in vitro transduction efficiency as described above. As expected, the number of assembled DNAse I-resistant genome copies increased over time (Figure 9A). We then rapidly affinity-purified crude vectors for early (days 1 and 2) and late (day 5) time points and measured their in vitro transduction efficiency in huh7 cells. The relative transduction efficiency of the vectors gradually decreased over time (Figure 9B). In terms of transgene expression per GC added to target cells, the day 5 vectors were only 40% of the day 1 material. This decrease in activity was also observed for the crude material, indicating a shift in molecular composition prior to purification (Figure 11). We observed a similar trend in AAV9 activity loss over the 5-day period, resulting in a roughly 40% decrease in vector potency (Figure 11I-K).

[0189] We next measured deamidation of the time-course samples by mass spectrometry. NG site deamidation progressed substantially over each interval, with an average of 25% deamidation on day 1 and over 60% of sites converted by day 5 (Figure 9C). Non-NG site deamidation generally progressed over the 5-day period, but at much lower levels and with less consistency from days 2 to 5 (Figure 9D). The data correlate endogenous vector deamidation with an early-time point decay in specific activity and highlight potential opportunities to capture more active vectors by shortening production cycles or identifying asparagine-stabilizing capsid mutations.

[0190] Note that the material used for mass spectrometry in Figures 2A-2E was at least 7 days post-transfection, as it took an additional 2 days for purification. Higher NG site deamidation (over 80%) indicates that deamidation likely continues at approximately the same rate after the expression period and during the recovery and purification process, until the NG sites are completely deamidated or the vector sample is frozen. Therefore, deamidation is primarily determined by the age of the vector and is not a process exclusive to or caused by the recovery and purification process. The much lower deamidation values ​​in the day 1 material compared to the day 5 material (both affinity purified) emphasize this point.

[0191] Stabilization of NG asparagine can improve vector performance Given the correlation between vector NG deamidation and loss of transduction efficiency, we reasoned that stabilizing NG amide by +1 site mutagenesis might improve vector function. We generated vectors on a small scale with AAV8 NG site mutants in which each +1 residue was individually converted to alanine or serine. The single +1 mutants were well tolerated in terms of vector assembly (Figure 10A) and transduction efficiency (Figure 10B). The G386 substitution (Aydemir), located near the previously defined "dead zone" on the capsid surface, significantly improved vector function. F, et al. J Virol July 2016;90(16):7196-204) was defective for in vitro transduction. The loss of function for the G386 mutant may indicate selection for a deamidated asparagine at N385. Alternatively, additional side chain bulk at the +1 position may adversely affect function independent of amide group stabilization. Single-site mutants did not significantly improve in vitro transduction despite dramatic stabilization of the adjacent asparagine (Table 2). Because in vitro and in vivo transduction activity may not match, we tested a subset of single-site +1 mutants for liver transduction in C57BL / 6 mice. Intravenous tail vein injections (n ​​= 3-5) were performed and luciferase expression was investigated by weekly imaging over a 2-week period (Figure 10C). The in vivo and in vitro transduction data were consistent within the relevant error of each assay (i.e., within the error margin). The G386 substitution was defective in transduction, but +1 site mutations at other positions were largely tolerated, transducing the liver at levels equal to, but not exceeding, wtAAV 8.

[0192] Because amide stabilization at any one NG site may be necessary but not sufficient for functional restoration, we next evaluated vector variants with combinations of alanine substitutions at the +1 site. We recombined all three AAV8 NG sites (N263, N514, and N540) for which the +1 alanine was highly functional. Some combinations, including the triple mutation G264A / G515A / G541A, assembled poorly and were dysfunctional for transduction. However, both pairwise combinations with N263 (G246A / G515A and G264A / G541A) improved in vitro transduction efficiency (2.0- and 2.6-fold over wtAAV8, respectively) without loss of titer (Figure 10D). Because these mutations result in at least two alterations (N-amide stabilization and +1 residue side chain substitution), these data do not conclusively link NG deamidation to loss of function. However, the data are consistent with a model established in time course studies in which NG site deamidation can affect in vitro transduction efficiency.

[0193] Functional asparagine substitution improves lot-to-lot reproducibility in vector manufacturing Another potentially problematic aspect of the reported vector deamidation profiles is the high lot-to-lot variability of deamidation at some positions. For wtAAV 8, this variability was most pronounced at N459 (observed deamidation ranged from 0% to 31%) and N499 (observed deamidation ranged from 0% to 53%). Variability in post-translational modifications is typically effectively avoided during biologic development by completely avoiding clones that exhibit this variability, carefully monitoring and controlling production strains and conditions, or by protein engineering of affected candidates.

[0194] Because we were unable to determine the production or processing factors contributing to N459 and N499 deamidation variability (Figure 2E), we sought functional amino acid substitutions at these positions. First, we individually evaluated small-scale vector preparations for conservative substitutions to glutamine at each position. Both N459Q and N499Q were efficiently incorporated into the vector and were comparable to the wtAAV8 reference for in vitro transduction efficiency (Figure 7A). Next, we produced the mutants on a larger scale and performed mass spectrometry analysis. Consistent with the observation of extremely rare glutamine deamidation, we observed selective and complete stabilization of glutamine amide at positions 459 or 499 in these mutants (data not shown). These mutant lots were evaluated in vivo for liver transduction after tail vein injection in C57BL / 6 mice as described above (Figure 7B and Figure 7C). The wT AAV8 vector lot used as a control in this experiment was 16.8% deamidated at N499, but no deamidation was detected at N459 (data not shown). Liver transduction of both mutants was comparable to wtAAV8 at day 14. This data demonstrates the potential of protein engineering approaches to address deamidation-related molecular variability in manufactured AAV vectors.

[0195] C. Consideration We independently identified and evaluated nonenzymatic deamidation of asparagine and glutamine residues on the AAV8 capsid using 2D gel electrophoresis, mass spectrometry, de novo protein modeling, and both in vitro and in vivo functional studies. Deamidation occurs in a wide variety of proteins and has been shown to significantly impact the activity of biologics, including antibody-based therapeutics (Nebija D et al. Int J Mol Sci 2014;15(4):6399-411) and peptide-based vaccines (Verma A et al. Clin Vaccine Immunol. 2016;23(5):396-402). Other viral proteins, such as the VP6 protein of rotavirus, have been shown to undergo deamidation events by mass spectrometry (Emslie KR et al. Funct Integr Genomics 2000;1(1):12-24).

[0196] The circumstances under which these deamidations occurred in AAV8 suggested they were the result of spontaneous, non-enzymatic events. Asparagine residues are known to be more extensively deamidated than glutamine residues, and amino acids downstream of asparagine substantially influence the rate of deamidation, with the N+1 glycine (i.e., NG) being the most efficiently deamidated. We observed striking confirmation of the role of the N+1 amino acid in AAV capsid deamidation in that all NGs present in VP1 were deamidated at levels greater than 75%, while deamidation did not consistently exceed 20% for any other asparagine or glutamine residue in the capsid. Virtually all NG motifs in AAV8 and AAV9 capsids (i.e., 7 / 9) were also present on the surface of the capsid, contained in HVR regions associated with conformational flexibility and thermal vibration. This is consistent with previous reports of NG motifs in other proteins located in regions where flexibility may be required for proper protein function, rather than in well-ordered structures such as alpha helices or beta sheets (Yan BX and Sun YQ J Biol Chem 1997;272(6):3190-4). The selection of a surface-exposed HVR NG motif further enhances the rate of deamidation by providing solvent accessibility and conformational flexibility, thereby facilitating the formation of the succinimidyl intermediate. As predicted, a less favorable environment leads to a much lower rate of deamidation.

[0197] An important question regarding the biology of AAV and its use as a vector is the functional consequence of these deamidations. Mutations in the capsid DNA that convert asparagine to aspartic acid allow for the evaluation of capsids in which all amino acids at a particular site are represented as aspartic acid. However, the direct consequences of the second site mutation, N+, confound the assessment. Other than potentially mutating a single residue, there is no easy strategy to prevent deamidation using mutagenesis. We studied limited variants in which the asparagine residue was converted to aspartic acid by mutagenesis. Functional analyses included capsid assembly and in vitro and in vivo transduction. The most substantial impact of mutagenesis on vector function was observed in those containing asparagines that were incompletely deamidated at baseline and not surface-exposed. Surprisingly, however, mutagenesis of the highly deamidated asparagine at 514 to aspartic acid had some effect on function. This result suggests that the presence of a corresponding amide residue may affect function. This may be due in part to the presence of a hydrogen-bonding interaction between N514 and D531 of another 3-fold related VP3 monomer (identified in the wtAAV8 crystal structure) that is lost upon conversion of this residue to aspartic acid after deamidation.

[0198] When assessing the impact of these deamidations on the development of novel therapeutics, it is important to better understand the factors that influence the degree of deamidation in AAV vectors. Incubating vectors under extreme conditions known to significantly accelerate deamidation kinetics had little effect. Combined with isotope incorporation studies, these results suggest that deamidation occurs during capsid assembly and is not an artifact of vector processing or mass spectrometry. Deamidation at NG sites is unlikely to substantially affect vector performance, as the reaction was virtually complete in all samples evaluated. However, initial functional studies suggest that residual amounts of non-deamidated asparagine can contribute to function. While there are concerns about sites with less complete deamidation, these were mostly related to inter-sample variability. One example is the asparagine at position 499, which showed a range of deamidation from 0% to 53%, with a mean value of 17%. Subtle differences in vector production conditions may contribute to this heterogeneity. The striking similarity of deamidation in AAV8 and AAV9 suggests that this is a property of this viral family as a whole.

[0199] In summary, we discovered substantial heterogeneity in the primary amino acid structure of AAV8 and AAV9 capsid proteins. These studies potentially impact the development of AAV as a vector in several ways. First, the actual amino acid sequence of the VP protein is not predicted by the corresponding DNA sequence. Second, aspects of the production method may lead to variations in deamidation and corresponding changes in vector function. Until we understand the factors affecting deamidation rates at non-NG sites and better understand their functional consequences, it may be necessary to include deamidation in the characterization of clinical-grade AAV vectors. While 2D gel electrophoresis can provide an overall assessment of net deamidation, mass spectrometry will be required to assess deamidation at specific residues.

[0200] Example 2: Deamidating AAV8 Triple Mutant (Clade E) The AAV8 triple mutant capsid was used to generate rAAV vectors. The predicted amino acid sequence of the VP1 protein of this capsid is provided herein in SEQ ID NO:9, and the nucleic acid sequence encoding the capsid is provided herein in SEQ ID NO:8. See also PCT application PCT / US17 / 27392, published as WO2017 / 180854.

[0201] The AAV8 triple mutant vector was evaluated for deamidation as described for AAV8 in Example 1. Highly deamidated residues were found at N57, N384, N498, N513, and N539. 10% to 40% deamidation was observed at N94, N254, and N255. Observed at N304, N409, and N516. [Table 15]

[0202] Example 3: Further deamidation studies Exemplary vectors were evaluated for deamidation as described for AAV8 and AAV9 in Example 1. AAV1 belongs to clade A, AAV7 belongs to clade D, and AAV3B, AAV5, AAVrh32 / 33, and AAV4 do not belong to any of clades A-F.

[0203] A. AAV1 Deamidation The AAV1 vector was evaluated for deamidation as described for AAV8 and AAV9 in Example 1. The results show that the vector contains four highly deamidated amino acids (N57, N383, N512, and N718) based on the numbering of the primary sequence of AAV1 VP1 reproduced in SEQ ID NO:1. [Table 16]

[0204] B. AAV3B deamidation AAV3B vectors were evaluated for deamidation as described for AAV8 and AAV9 in Example 1. Referring to the AAV3B numbering, high levels of deamidation were observed at four asparagine residues: N57, N382, N512, and N718. These numbers are based on AAV3B VP1, as reproduced in SEQ ID NO:2. [Table 17]

[0205] C.AVV5 deamidation AAV5 vectors were evaluated for deamidation as described in Example 1 for AAV8 and AAV9. High levels of deamidation were observed at residues N56, N347, N347, and N509. Approximately 1% to approximately 35% deamidation was observed at positions N34, N112, N213, N243, N292, N325, N400, N421, N442, N459, and N691. These numbers are based on AAV5 VP1, as reproduced in SEQ ID NO:3. [Table 18]

[0206] D. AAV7 Deamidation AAV7 vectors were evaluated for deamidation as described in Example 1 for AAV8 and AAV9. High levels of deamidation were observed at N41, N57, N384, and N514. Deamidation at rates ranging from 1% to 25% was observed at N66, N224, N228, N304, N499, N517, N705, and N736. These numbers are based on AAV7 VP1, reproduced in SEQ ID NO:4. [Table 19]

[0207] E. AAVrh32.33 deamidation The AAVrh32.33 vector was evaluated for deamidation as described in Example 1 for AAV8 and AAV9. High levels of deamidation were observed at positions N57, N264, N292, and N318. Between 1 and 45% deamidation was observed at positions N14, N113, Q210, N247, Q310, N383, N400, N470, N510, and N701. These numbers are based on the rh32.33 AAV VP1, reproduced in SEQ ID NO:5. [Table 20]

[0208] F. AAV4 Deamidation AAV4 was evaluated as previously described. High levels of deamidation were observed at positions 56 and 264. Other positions with high levels of deamidation may include positions 318 and 546. [Table 21]

[0209] Trypsin and chymotrypsin preparations are reported separately. However, certain residues are lost by trypsin or chymotrypsin based on the sequence and peptides obtained. When a residue is observed in both preparations, the deamidation is consistent and should not deviate significantly from the average.

[0210] Example 4: Mapping adeno-associated virus 9-specific neutralizing epitopes In this study, we attempted to identify neutralizing epitopes in AAV9 that have not yet been evaluated by this epitope mapping approach. Importantly, AAV9 is currently administered intravenously in the clinic for several cardiac, musculoskeletal, and central nervous system indications (Bish LT, et al. Hum Gene Ther. 2008;19(12):1359-68; Foust KD, et al. Nature Biotechnology. 2009;27(1):59-65; Kornegay JN, et al. Molecular Therapy. 2010;18(8):1501-8), particularly spinal muscular atrophy (Mendell JR, et al. N Engl J Med. 2017;377(18):1713-22). Here, we report the highest-resolution AAV-Ab complex reconstructed to date: a 4.2 Å structure of AAV9 in complex with the potent NAb PAV9.1. Through the use of serotype swapping, alanine substitutions, and additional point mutations, we validated the epitope of PAV9.1 and demonstrated that the resulting mutants mimic PAV9. We demonstrated the ability to significantly disrupt PAV9.1 binding and neutralization. However, when the mutants were tested against a panel of polyclonal samples from various sources, this effect on both the binding and neutralization capacities of PAV9.1 was significantly reduced or not observed. This result suggests that in some situations, this epitope may play a role in neutralizing AAV transduction, but targeted mutation of a broader range of neutralizing epitopes will be necessary to engineer novel capsids that can circumvent the repertoire of NAbs involved in blocking AAV transduction.

[0211] A. Materials and Methods 1. Hybridoma Generation Balb / c mice received up to five immunizations with AAV9 vectors. Spleen cells were collected and fused. ProMab Biotechnologies, Inc. (Richmond, CA) generated clonal supernatants according to the company's standard custom mouse monoclonal antibody hybridoma development protocol. Thirty supernatants were screened for AAV9 reactivity by ELISA and for their ability to neutralize AAV9 by NAb assay. After screening at a concentration of 3 mg / mL, purified PAV9.1 mAb was obtained.

[0212] 2. AAV Capsid ELISA Corning polystyrene high-binding microplates were coated with 1e9 GC / well AAV diluted in phosphate-buffered saline (PBS) and incubated for 4 min. o The plates were then incubated at 37°C overnight. After discarding the coating solution, the plates were blocked with 3% bovine serum albumin (BSA) in PBS for 2 hours at room temperature and washed three times with 300 μL PBS + 0.05% Tween. Hybridoma supernatants, purified mAbs, serum, or plasma (diluted in 0.75% BSA in PBS) were then incubated for 1 hour at 37°C and washed three times with 300 μL PBS + 0.05% Tween. Next, 1:10,000 goat anti-mouse IgG HRP (diluted in 0.75% BSA in PBS; catalog number 31430; Thermo Scientific) was incubated for 1 hour at 37°C. Mouse samples were detected using ELISA kits (Fisher Scientific, Waltham, MA) followed by triple washes of 300 μL PBS + 0.05% Tween. Human and nonhuman primate samples were then detected using goat anti-human IgG biotin-SP (catalog no. 109-065-098, Jackson ImmunoResearch Inc., West Grove, PA) at 1:10,000 (diluted in PBS) for 1 hour at room temperature, followed by triple washes of 300 μL PBS + 0.05% Tween, and streptavidin (catalog no. 016-000-084, Jackson ImmunoResearch Inc., West Grove, PA) at 1:30,000 (diluted in PBS) for 1 hour at room temperature, followed by three washes of 300 μL PBS + 0.05% Tween. All ELISAs were developed using tetramethylbenzidine.

[0213] 3. Neutralizing Antibody Assay NAb assays were performed as previously described with some modifications (Calcedo R, et al. J Infect Dis. 2009;199(3):381-90). HEK293 cells were used, seeded at a density of 1e5 cells / well in black-walled, clear-bottom, polylysine-coated plates (catalog no. 08-774-256, Fisher Scientific Company, Hampton, NH). A multiplicity of infection of 90 wtAd5 / cell was used, utilizing a working solution of AAV9.CMV.LacZ vector at 4e10 GC / mL to achieve a final concentration of 2e9 GC / well. Bioluminescence was measured using a SpectraMax M3 (Molecular Devices, Sunnyvale, CA) according to the manufacturer's protocol. For any given sample, NAb titers were defined as the last dilution at which AAV transduction was reduced by more than 50% in the presence of the sample compared to WT.AAV transduction in the presence of an untreated control. HEK293 transduction experiments were performed as described above. However, the neutralizing serum was withheld.

[0214] 4. Fab Generation and AAV-Fab Conjugation PAV9.1 Fab (0.211 mg / mL) was generated using the Pierce Fab Preparation Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The PAV9.1 Fab was then complexed with the AAV9 vector at a ratio of 600 Fab:1 AAV9 capsid (or 10 Fab:1 potential binding site) for 30 minutes at room temperature.

[0215] 5. Cryo-EM Sample Preparation, Data Acquisition, and Complex Reconstruction Sample preparation: 3 μL of PAV9.1-AAV9 complexes were applied to freshly cleaned glow-discharged holey carbon grids. After blotting with Whatman #1 filter paper for 3–4 seconds at 22 °C and 95% relative humidity, the grids were rapidly frozen in liquid ethane slush using a Vitrobot Mark IV (FEI). Next, the grids were frozen at 22 °C and 95% relative humidity. o A single blot of 3–4 s was applied with Whatman filter paper (C). After freezing, the grids were stored in liquid nitrogen. The grids were then transferred to an FEI Talos Arctica electron microscope operated at 200 kV and equipped with a Gatan K2 Summit direct electron detection camera (Gatan, Pleasanton, USA).

[0216] Data acquisition: Data were acquired using SerialEM software (Mastronarde DN. J Struct Biol. 2005;152(1):36-51). Images were captured at a nominal magnification of 22,000x (corresponding to a calibrated pixel size of 0.944 Å) and a dose rate of 2.21 electrons / square Å / s with a defocus range of 1.0-2.0 μm (Rohou A. and Grigorieff N. Struct Biol. 2015;192(2):216-21). For each exposure, a 60-frame dose-split movie stack was recorded in super-resolution mode for a total of 12 seconds. Movie frames were aligned using the "Align Frames" program within the IMOD software package (Kremer JR, et al. J Struct Biol. 1996;116(1):71-6).

[0217] Data collection and processing: All particle images were manually extracted from each micrograph and processed using the e2boxer program available in the EMAN2 suite (Tang G, et al. J Struct Biol. 2007;157(1):38-46). The boxed particles were then transferred to the AUTO3DEM program for freeze-reconstruction, resulting in an initial low-resolution model (30 Å) based on 150 particle images (Yan X, et al. J Struct Biol. 2007;157(1):73-82). The program employed a random model generation procedure and applied strict 60 non-crystallographic symmetry axes. This low-resolution reconstructed model map was useful for determining particle origins and refining the contrast transfer function of all images using AUTO3DEM. To improve the quality of the reconstructed maps, temperature coefficient correction was applied, and the maps were visualized with the graphics programs Coot and Chimera (Pettersen EF, et al. J Comput Chem. 2004;25(13):1605-12, Emsley P and Cowtan K. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2126-32). A temperature coefficient 150-corrected map was used for docking and interpretation of the model. A total of 3,022 box particles were extracted from 1,100 micrographs, ultimately generating a 4.2 Å resolution reconstructed map with a Fourier shell correlation of 0.15. The AAV9-60mer model was generated using the VIPER database, applying a strict icosahedral symmetry axis (T=1) (Carrillo-Tripp M, et al. Nucleic Acids Res. 2009;37(Database issue):D436-4). 2). A 60-mer copy of the AAV9 capsid was docked to the cryo-reconstructed electron density map using the FIT function in the Chimera program. This produced a correlation coefficient of 0.9. To improve accuracy, the docking models in Coot and Chimera were visualized and adjusted. Antibody models were generated using ABodyBuilder, then docked and manually adjusted to the cryo-reconstructed density using Chimera (Leem J, et al. (e.g., DeLano WL. PyMOL: An Open-Source Molecular Graphics Tool. 2002; Vol. 40: 82-92). A two-dimensional representation of the roadmap was created using the RIVEM program (Xiao C and Rossmann MG. J Struct Biol. 2007. 158(2): 182-7). The model was then visualized for interpretation of the AAV9 and antibody binding regions. All figures were generated using the Chimera and PyMOL programs. A two-dimensional representation of the roadmap was created using the RIVEM program (DeLano WL. PyMOL: An Open-Source Molecular Graphics Tool. 2002; Vol. 40: 82-92). A two-dimensional representation of the roadmap was created using the RIVEM program (Xiao C and Rossmann MG. J Struct Biol. 2007. 158(2): 182-7).

[0218] 6. AAV9-PAV9.1 Mutant Transformer Plasmid Construction For AAV9 capsid mutagenesis, we used the in-house trans-plasmid construct pAAV2 / 9 (AAV2 rep / AAV9 cap). All capsid mutants were constructed using the Quikchange Lightning Mutagenesis Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions.

[0219] 7. Vector Production AAV9.CMV.LacZ.bGH and AAV9 mutant vectors were produced by triple transfection in HEK293 cells followed by iodixanol gradient purification as previously described (Lock M, et al. Hum Gene Ther. 2010;21(10):1259-71). The University of Pennsylvania Vector Core titered the vectors using quantitative PCR (qPCR) against bGH polyA as previously described (Lock M, et al. Hum Gene Ther. 2010;21(10):1259-71).

[0220] 8. Determine the EC50 of PAV9.1 mAb and polyclonal serum / plasma. Capsid capture ELISAs were performed using either AAV9.WT or AAV9 mutant vectors as described above. EC50 values ​​were calculated using GraphPad Prism. Briefly, PAV9.1 mAb concentrations were log-transformed in mg / mL and plotted on the x-axis. IgG concentrations in mouse plasma were defined as 5 mg / mL (Mink JG. Serum immunoglobulin levels and immunoglobulin heterogeneity in the mouse. Diss. Erasmus MC. 1980), and IgG concentrations in non-human primate and human serum were defined as 10 mg / mL (Gonzalez-Quintela A, et al. Clinical and Experimental Immunology. 2008;151(1):42-50). Plasma / serum concentrations (μg / mL) were log-transformed and plotted on the x-axis. The maximum absorbance achieved for each mutant was defined, and absorbance was normalized to 100% and plotted on the y-axis. Dose-response curves (antibody binding) were then generated using the "Log(agonist) vs. normalized response - variable slope" function in GraphPad Prism. EC50 was calculated for mAb, polyclonal serum, or polyclonal plasma.

[0221] 9. Animal research The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and was carried out in accordance with its standards. Male C57BL / 6 mice (n = 3) were transfected with either 1e11GC / mouse AAV9.CMV.LacZ.bGH or 1e11GC / mouse AAV9.CMV.LacZ.bGH carrying the same transgene cassette. The animals received an intravenous injection of the AAV9 mutant vector into the tail vein. 14 days after receiving the vector, the animals were sacrificed. Organs from each animal were sectioned and either flash-frozen on dry ice for biodistribution or embedded in optimal cutting temperature compound, frozen for subsequent sectioning, and stained for β-gal activity.

[0222] 10. Biodistribution Analysis DNA was extracted from tissues of interest using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Tissues were analyzed for vector GC expression by qPCR for the bGH polyadenylation signal as previously described (Chen SJ, et al. Hum Gene Ther Clin Dev. 2013;24(4):154-60).

[0223] 11.β-gal activation staining Frozen sections were fixed with 0.5% glutaraldehyde in PBS for 10 minutes at 4°C and then stained for β-gal activity. After washing with PBS, sections were incubated in 1 mg / mM X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in 20 mM potassium ferrocyanide, 20 mM potassium ferricyanide, and 2 mM MgCl2 in PBS (pH 7.3), and the tissue was kept overnight at 37°C. Sections were counterstained with Nuclear Fast Red (Vector Laboratories), dehydrated using ethanol and xylene, and then coverslipped.

[0224] B. Results 1. NAb PAV9.1 is potent and specific for AAV9 We first sought to identify novel, potent anti-AAV9 NAbs for epitope mapping. A panel of 30 hybridoma clones was screened for AAV reactivity by enzyme-linked immunosorbent assay (ELISA) against multiple serotypes and for AAV9 neutralization by NAb assay. From this panel, we selected the monoclonal antibody PAV9.1 for its specificity for AAV9 (Figure 12A). PAV9.1 recognized only intact capsids by ELISA (Figure 12A) and did not recognize AAV by Western blot (data not shown), suggesting that PAV9.1 recognizes a conformational epitope on the capsid surface. This contrasts with the remaining clones, which bound more broadly to the panel of AAVs included in the screen and also recognized AAV by Western blot (data not shown). In the NAb assay, the purified PAV9.1 mAb showed an effective NAb titer of 1:163,840, indicating that this novel anti-AAV9 antibody is a potent neutralizer of AAV9. Again, this is in contrast to other clones screened by NAb assay, none of which were able to neutralize AAV transduction.

[0225] 2. Cryoreconstitution of AAV9 in complex with PAV9.1 After complexation of AAV9 with the PAV9.1 antigen-binding fragment (Fab), 1,100 images were captured, 3,022 particles were boxed, and a 4.2 Å reconstruction of the complex was generated using AUTO3DEM. Fab density was observed extending from the 3-fold axis formed by HVRs IV, V, and VII, with Fab electron density vertically centered to decorate the inner surface of the 3-fold protrusions (Figures 13A and 13B). This region is primarily composed of charged residues, promoting strong electrostatic interactions between 3-fold-associated VP monomers and with the receptor and mAb. A single Fab molecule was bound and extended at each 3-fold axis across two of the three protrusions, blocking binding of additional Fab molecules at these sites due to steric hindrance (Figure 13C). The regions of the PAV9.1 Fab complementarity-determining regions (CDRs) that contact the 3-fold protrusions had an average density of 2.5 sigma, comparable to the densities reported for other AAV-Fab reconstructions. At approximately the 0.8 sigma level, we observed PAV9.1 Fab constant region density, or approximately one-third of the density observed in the PAV9.1 CDR contact region, corresponding to a single Fab occupancy per 3-fold axis. The PAV9.1 Fab CDR directly interacted with residues 496-NNN-498 (HVR V) and 588-QAQAQT-593 (HVR VIII) (Figures 13C and 13D). PAV9.1 binding further excluded G455 and Q456 (HVR IV), T494, Q495, and E500 (HVR V), as well as N583, H584, ​​S586, and A587 (HVR VIII), which are not involved in electrostatic interactions with PAV9.1 but may provide structural stability to this region of the capsid after Fab binding (Table 3). The CDRs of the heavy chain interacted with HVR V, whereas the CDRs of the light chain interacted with HVR VIII of the same VP3 monomer (Fig. 13C). [Table 22]

[0226] Based on the PAV9.1 footprint (Figure 13D, Table 3), two sets of five residues were selected for mutagenesis focused on epitope validation and escape mutation design: 586-SAQAQ-590 and 494-TQNNN-498. Residues 586-SAQAQ-590 were chosen because this site contains a high degree of sequence diversity (Figure 12B). The selected motif includes residues identified by the reconstruction as directly interacting with PAV9.1 and residues identified as excluded, allowing for investigation of the junction between the binding and excluded residues. These residues are also involved in the neutralization epitopes of AAV1, AAV2, and AAV8, allowing comparison of the AAV9 epitope residues with those previously published (Tseng YS and Agbandje-McKenna M. Front Immunol. 2014;5:9). Finally, because this motif has more limited interactions with regions that contribute to the structural integrity of the capsid, restricting HVR VIII mutagenesis to these five residues increased the likelihood that the capsid would tolerate larger mutations. Despite PAV9.1 being specific to AAV9, the HVR V motif 496-NNN-498, which we identified as interacting with PAV9.1, is highly conserved across serotypes (Figure 12B). However, unpublished phage display studies (data not shown) suggested the involvement of an asparagine-rich motif in the PAV9.1 epitope, and therefore, we selected this motif for mutagenesis. We also added residues 494-TQ-495 to re-examine the junction between the binding and exclusion residues (Tseng YS and Agbandje-McKenna M. Front Immunol. 2014;5:9).

[0227] 3. Epitope-based mutations significantly reduce AAV9-PAV9.1 binding First, we used site-directed mutagenesis to generate the 586-SAQAQ-590 serotype swap mutant. Based on the knowledge that PAV9.1 specifically recognizes AAV9 and that the amino acid sequence and structural shape at this position vary significantly among AAV serotypes, we selected a complete swap with the corresponding residue from representative serotypes from clade B (AAV2), clade C (AAV3B), and clade D / E (AAV8 / rh10) (Table 4). [Table 23]

[0228] In doing so, we hoped to maximize the potential for efficient capsid assembly while also maximizing natural variation at this site. We generated two additional mutants, AAV9.AAQAA (more convergent than AAV9.QQNAA) and AAV9.RGHRE (more divergent than AAV9.RGNRQ), to determine (1) the minimum mutations required to disrupt PAV9.1 interactions and (2) the maximum disruption that could be introduced. The AAV9.AAQAA, AAV9.QQNAA, and AAV9.SSNTA mutants produced vector titers comparable to AAV9.WT, whereas titers of AAV9.RGNRQ and AAV9.RGHRE were reduced 2-3-fold compared to AAV9.WT (data not shown). The binding of each mAb to each mutant capsid was determined by capture ELISA compared to AAV9.WT (Figure 14A). The EC50 for each swap mutant, or the concentration of PAV9.1 mAb required to reach half-maximal binding, was significantly increased compared to the EC50 for AAV9.WT (indicating reduced capsid binding). This result validates the epitope mapping results and indicates that residues 586-SAQAQ-590 are involved in the AAV9-PAV9.1 interaction. The EC50 increase ranged from 45-fold (AAV9.AAQAA) to nearly 300-fold (AAV9.RGHRE) (Table 5), and the EC50 increase directly correlates with the degree of sequence divergence from AAV9 at this position. One exception is AAV9.RGNRQ, which shares Q590 with AAV9, potentially contributing to stronger PAV9.1 binding than predicted by sequence analysis. [Table 24]

[0229] Because the S586A and Q590A mutations in AAV9.AAQAA were sufficient to disrupt AAV9 binding to PAV9.1, we next determined the minimal change required to cause this disruption. Point mutations were introduced at one of these positions by either alanine substitution or more conservative substitutions (S → T or Q → N). Mutation of S586 to alanine or threonine did not significantly reduce PAV9.1 binding, whereas a single mutation of Q590 to alanine or asparagine was sufficient to disrupt capsid recognition by PAV9.1 (Figure 14C). This result indicates that position 590 is important for PAV9.1 recognition of the AAV9 capsid.

[0230] Next, we used the same mutagenesis strategy to investigate the 494-TQNNN-498 motif in the HVR V, which is contained in the PAV9.1 epitope, and mutated the set of residues to evolutionarily conserved amino acids or alanine alone. Because 496-NNN-498 is conserved across all serotypes tested, we used only alanine substitutions for this extension of residues, and mutated 494-TQ-495 to AA, GQ, and TD to represent the naturally occurring diversity at this site. Despite the specificity of PAV9.1 for AAV9 and the diversity at this position, AAV9.GQNNN, AAV9.TDNNN, and AAV9.AANNN did not increase the EC50 of PAV9.1 against AAV (Figure 14B). This supports the conclusion from the cryo-reconstruction map that the 494-TQ-495 site is not involved in the PAV9.1 epitope. However, the AAV9.TQAAA mutation increased the PAV9.1 EC50 by 15-fold, indicating that the 496-NNN-498 motif, despite being a conserved motif, plays an important role in AAV9-specific binding of PAV9.1. Finally, we generated combination mutants from HVR V and minimal HVR VIII mutations (AAV9.TQAAA / SAQAN, AAV9.TQAAA / SAQAA). The PAV9.1 EC50 values ​​of these combination mutants demonstrate that the effects of altering the PAV9.1 epitope motif are additive (Figure 14B and Figure 14E).

[0231] 4. Epitope-based mutations modulate AAV9 transduction To assess the ability of the novel AAV9 mutants to evade NAb while maintaining the properties of AAV9.WT, we first evaluated in vitro and in vivo transduction. Most of the mutations that resulted in reduced PAV9.1 binding also reduced transduction efficiency in HEK293 cells, except for AAV9.RGNRQ, which improved vector transduction by 2.3-fold (Figure 15A). This improvement may have been due to the introduction of R586 and R589 (R585 and R588 according to AAV2 VP1 numbering), two residues involved in heparin recognition by AAV2, due to the high likelihood that these heparin-binding motifs are involved (Ellis BL, et al., Virol. J. 2013;10(1):74), significantly outperforms AAV9 in vitro in most cell lines. However, AAV9.RGHRE, which shares R586 and R589 with AAV9.RGNRQ, did not display AAV2-like transduction efficiency, suggesting the involvement of other factors. AAV9.AAQAA showed the greatest reduction in transduction efficiency, indicating that S586 and / or Q590 are essential residues for AAV9 transduction in vitro.

[0232] 5. Epitope-based mutations abolish PAV9.1 neutralization Next, we investigated the effect of mutations on the neutralization titer of PAV9.1. The AAV9.AANNN mutation, which does not affect PAV9.1 binding, did not affect neutralization titer (Figures 15B and 15I). However, all mutant vectors that increased the PAV9.1 EC50 reduced the effective neutralization titer of PAV9.1. AAV9.RGHRE dramatically increased the EC50 by nearly 300-fold and reduced the NAb titer of PAV9.1 by at least 2,048-fold (from 1:163,840 to <1:80, the lowest dilution tested) (Figures 15C–15K). Mutant vectors that only modestly increased the EC50, such as AAV9.SAQAN, reduced the effective NAb titer of PAV9.1 to a lesser extent (Figure 15L). Overall, we observed a strong correlation between reduced PAV9.1 binding, as measured by EC50, and reduced effective NAb titers (Figure 16). The notable exception was again AAV9.RGNRQ, which, despite being the fourth effective mutation that reduced PAV9.1 binding, only reduced NAb titers by 8-fold (a second reduction).

[0233] 6. The PAV9.1 epitope is important for AAV9 liver tropism To evaluate the viability of these mutants as AAV9-like gene therapy vectors, C57BL / 6 mice were intravenously injected with 1e11 genome copies (GC) / mouse of AAV9.WT.CMV.LacZ or AAV9 mutant vectors (n=3 per group) with reduced PAV9.1 activity. Biodistribution of tissue samples on day 14 showed reduced liver transduction for all mutants. AAV9.QQNAA performed most similarly to AAV9.WT, with a 17-fold reduction in GC / µg DNA, while AAV9.RGHRE transduced the liver least efficiently, with a 1,110-fold reduction in GC / µg DNA (Figure 17A). However, in other organs, such as the heart and brain, the majority of the mutants maintained near AAV9.WT levels of transduction, with the exception of the AAV2-like mutants AAV9.RGNRQ and AAV9.RGHRE. Although these differences in tissue GC were not statistically significant, the observed trends suggest that these residues are important for AAV9 liver tropism but play a lesser role in transduction of other tissues, as most mutants exhibited a "liver-nontargeting" phenotype. These results were further reflected in beta-galactosidase (β-gal) expression in the liver and heart; liver β-gal activity was highest in animals receiving AAV9.WT, while cardiac β-gal activity was similar for AAV9.WT and most mutants (except the AAV2-like mutant) (Figures 17B and 17C).

[0234] For a representative subset of AAV9 mutant vectors, these experiments were repeated at a 10-fold higher dose (1e12 GC / mouse), at which the difference in transduction did not reach significance. Although the tissue tropism was not significant, the trend in tissue tropism was consistent with that observed at lower doses, particularly for heart and muscle samples (Figure 17D). Again, these results were reflected in β-gal activity in liver, heart, and muscle tissue sections (Figures 17E-G).

[0235] 7. Epitope-based mutations in AAV9 do not significantly affect binding or neutralization by polyclonal plasma or serum Next, we evaluated the ability of mutant vectors based on the PAV9.1 epitope to evade binding and neutralization by polyclonal plasma or serum. First, we utilized plasma from C57BL / 6 mice previously intravenously injected with AAV9.WT (7.5e8 or 7.5e9 GC / mouse, n=6 per group). We determined the plasma dilution required to reach half-maximal binding. Plasma binding with mutant vectors from low-dose mice was nearly indistinguishable from that with AAV9.WT (Figures 18A-C). In contrast, we observed significant differences in the EC50 of plasma from high-dose mice for a subset of mutants, particularly AAV9.RGNRQ, relative to the EC50 of AAV9.WT (Figures 18B-D). Despite an average 2-fold increase in the EC50 of high-dose mouse plasma for AAV9.RGNRQ, we did not observe a decrease in effective NAb titers in plasma with this mutation (data not shown).

[0236] To determine whether this trend in EC50 increases applies to non-human primate samples, we obtained serum from a panel of six macaques that received either the AAV9 vector or a novel vector closely related to AAV9 that shares the same VP3 sequence (a two-amino acid difference in the nonstructural VP1 region). Prior to administration, we confirmed that the macaques had NAb antibody titers to AAV9 greater than 1:5 (defined as NAb-negative). Although we observed some variation in the EC50 values ​​of each animal's serum for the mutant vectors compared to the EC50 of AAV9.WT, no clear trend of increased or decreased binding emerged based on mutation identity (Figures 19A and 19C). When testing serum from macaques with pre-existing NAb titers to AAV9 (due to previous AAV infection), we observed little change in the EC50 values ​​of the serum for the panel of AAV9 mutants (Figures 19B and 19D). This is in stark contrast to the variation seen in the EC50 of injected sera, suggesting fundamental differences between the relevant anti-AAV epitope repertoires of sera generated in response to AAV infection and AAV vector administration. Additionally, the increase in the EC50 of AAV9.RGNRQ-injected non-human primate sera did not decrease the effective NAb titers of AAV9.RGNRQ-injected sera (data not shown).

[0237] Finally, NAb-positive serum samples from four normal human donors were evaluated for binding to AAV9.WT and mutant vectors. As with the uninjected NAb-positive non-human primate serum samples, all four NAb-positive normal human donor samples showed minimal variation in EC50 values ​​for AAV9 mutant and WT vectors (Figures 20A-B). As expected, the lack of change in EC50 values ​​for the mutant vectors indicated a lack of reduction in serum NAb titers against the AAV9 mutant vectors (data not shown).

[0238] C. Consideration Here, we report the cryo-reconstitution of AAV9 in complex with the highly potent and specific mAb PAV9.1. The epitope determined for PAV9.1 largely overlaps with the epitope regions of other AAV NAbs isolated from murine hybridomas: ADK8 (AAV8; 586-LQQQNT-591), E4E (AAV1; 492-TKTDNNN-498), 5H7 (AAV1; 496-NNNS-499, 588-STDPATGD-595), and C37 (AAV2; 492-SADNNNS-498, 585-RGNRQ-589) (Gurda BL, et al. J Virol. 2012;86(15):7739-51, Gurda BL, et al. J Virol. 2013;8 7(16):9111-24, Tseng YS, et al. J Virol. 2015;89(3):1794-1808). Thus, despite the large degree of sequence and structural variation between serotypes in HVRs V and VIII, this finding suggests that the three-fold protrusion may be a significant site of AAV9 neutralization, as in other serotypes. Thus, previous findings regarding the repertoire of NAbs directed against other AAV capsids may be applicable to AAV9. Although various mapped neutralizing epitopes show overlap, the binding angles and orientations of NAbs differ significantly. Upon binding to AAV9, PAV9.1 extends toward the center of symmetry of the three-fold axis, sterically restricting occupancy to 20 Fab particles. In contrast, mAbs raised against other serotypes bind above or outward from the three-fold axis, allowing for higher occupancy. Studies have identified both HVR V and VIII as shared antigenic regions across serotypes, including AAV2 (complexed with C37B, 11 Å), AAV8 (complexed with ADK8, 18.7 Å), and AAV1 (complexed with 5H7, 23 Å), with the binding footprint of PAV9.1 most similar to that of AAV9 (Gurda BL, et al. J Virol. 2012;86(15):7739-51; Gurda BL, et al. J Virol. 2013;87(16):9111-24; Tseng YS, et al. J Virol. 2015;89(3):1794-1808). Thus, the structure reported here is similar to previously reported lower-resolution structures for other AAV serotypes.

[0239] HVR VIII serotype swapping conferred varying degrees of binding and neutralization evasion to their corresponding mutant vectors. Swapping this region with the AAV2-based RGHRE motif, the most divergent mutation from the WT AAV9 sequence, abolished PAV9.1 neutralization at all dilutions tested. Thus, manipulating only five amino acids in the capsid can evade monoclonal Nab. Indeed, the minimal change required to significantly reduce PAV9.1 activity was a single amino acid substitution, and even conserved amino acids led to a loss of both binding and neutralization. Mutation of the NNN motif in HVR V, despite its high conservation across serotypes, reduced the ability of PAV9.1 to bind and neutralize AAV9, indicating that it is also an integral part of the PAV9.1 epitope.

[0240] We observed a strong correlation between the reduced binding of PAV9.1 to a given AAV9 mutation and its ability to block transduction in vitro, suggesting that the relative strength of NAb antibodies against AAV correlates with the neutralizing capacity of the NAb. However, data from our laboratory and others suggest that the binding antibody titer to AAV is not necessarily a good predictor of an individual's NAb titer, as some individuals have moderate binding titers to AAV but are NAb-negative (Falese L, et al. Gene Ther. 2017;24(12):768-78; Huttner NA, et al. Gene Ther. 2003;10(26):2139-47) (unpublished data). Despite these findings, the exclusion criteria for some clinical trials include not only NAb titer but also binding titer (George LA, et al. Blood. 2017;130(Suppl 1):604; Mendell JR, et al. N Engl J Med. 2017;377(18):1713-22). Therefore, epitope mapping studies are important to identify the characteristics of binding epitopes and determine whether they share any commonalities with neutralizing epitopes. Shared motifs suggest that binding strength, rather than interaction with specific residues, plays a major role in AAV neutralization, thus allowing researchers to focus solely on reducing NAb binding. However, distinct motifs suggest that neutralization is a function of binding location rather than binding strength, suggesting that researchers should focus on eliminating NAb binding to these unique regions.

[0241] Mutations in the AAV9 vector significantly reduced binding and neutralization by purified monoclonal PAV9.1 antibodies, but these mutations did not significantly prevent binding or neutralization by polyclonal antibodies derived from serum or plasma of mice, macaques, or human donors previously exposed to AAV. Most notably, plasma from mice receiving higher intravenous doses of AAV9 vector bound the RGNRQ mutant approximately two-fold less efficiently than the WT.AAV9 vector, a change much milder than the 50-fold reduction observed with the PAV9.1 mAb. Mutations in QQNAA, SSNTA, and RGHRE had a greater effect on PAV9.1 binding and neutralization than mutations in RGNRQ, yet polyclonal plasma bound these mutants in the same manner as WT.AAV9. These results suggest that the 586-SAQAQ-590 motif is a potent neutralizing epitope and that mutations in this region can block PAV9.1 activity, but in vitro activity against mAbs does not predict activity against polyclonal antibodies. Perhaps surprisingly, the RGNRQ mutation efficiently blocked AAV9 antibody binding by using the three-fold protrusion. This result clearly demonstrates that not all mutations function equally well against polyclonal responses, and that a larger repertoire of antibodies utilizes this region for binding.

[0242] Despite the reduction in polyclonal binding, the RGNRQ mutant vector did not circumvent the polyclonal NAb responses generated by these mice in response to vector administration. As expected, mutants that did not reduce binding to polyclonal plasma also did not circumvent neutralization. Considering that a nearly 100-fold increase in the EC50 of PAV9.1 against RGNRQ compared to WT.AAV9 resulted in only an 8-fold decrease in PAV9.1 neutralization titer, it was not surprising that a 2-fold increase in the EC50 of polyclonal plasma against RGNRQ did not reduce neutralization titer. While the study indicates that the majority of mapped AAV epitopes are located on the 3-fold axis and that HVR VIII is involved in mapped epitopes for most serotype-specific NAbs, we were surprised that none of the mutations tested in this region dramatically affected polyclonal activity. (Note that due to the small total number of mapped epitopes and the uncertainty about the precise screening and selection methods of some studies, the mapped epitopes may not represent the complete repertoire.)

[0243] Tse and colleagues recently used a library approach to combine epitopes from three distinct NAb sequences identified against AAV1, generating a novel AAV1-based capsid with over 20 amino acid changes from the parent AAV1. This capsid was able to evade not only anti-AAV1 monoclonal NAb sequences but also polyclonal sequences from AAV-exposed normal human donors, as well as polyclonal sequences from AAV vector-injected mice and non-human primates (Tse LV, et al. Proc Natl Acad Sci USA. 2017;114(24),E4812-21). This suggests that neutralizing epitopes may overlap after vector exposure and viral infection, but this repertoire is slightly diverse. In other words, the total number of residues requiring modification to confer AAV binding and neutralization evasion is more extensive than previously thought. Combinatorial and high-throughput approaches may be necessary to engineer novel capsids that can address both scenarios.

[0244] This study investigated whether vectors engineered to evade pre-existing NAb responses from prior AAV infection would function in a rechallenge setting. Polyclonal samples in which PAV9.1-based AAV9 mutant vectors showed minimal evasion were obtained from sources that received AAV vectors, but not from sources previously infected with AAV. While injected samples exhibited modestly varying binding curves to the panel of AAV9 mutants, binding curves generated by vector-naive but virus-exposed sources were similar to those for WT.AAV9. These differences may be due to differences in AAV antibodies generated in response to vector administration or infection. Highlighting fundamental differences between repertoires.

[0245] Historically, naive subjects injected with AAV vectors generate NAb responses that are specific to or restricted to closely related serotypes of the administered vector (Flotte TR, et al. Hum Gene Ther. 2011;22(10):1239-47) (unpublished data). Most macaque studies and gene therapy clinical trials have shown similar results (Greig JA, et al. Vaccine. 2016;34(50):6323-29, Greig JA, et al. Hum Gene Ther Clin Dev. 2017;28(1):39-50) (unpublished data). In stark contrast, subjects with pre-existing antibodies to one AAV serotype are almost always seropositive and have NAbs to most other serotypes, even distantly related ones (Calcedo R and Wilson JM. Hum Gene Ther Clin Dev. 2016;27(2):79-82; Flotte TR, et al. Hum Gene Ther. 2011;22(10):1239-47; Harrington EA, et al. Hum Gene Ther. 2016;27(5):345-53 (unpublished data). To date, all newly mapped AAV mAbs are specific for individual serotypes and cross-react only with closely related serotypes (e.g., 5H7, which binds both AAV1 and AAV6), and previously isolated neutralizing AAV mAbs do not recapitulate the broad responses typically seen after AAV infection (Gurda BL, et al. J Virol. 2013;87(16):9111-24). Therefore, further studies are needed to identify motifs containing broadly neutralizing epitopes relevant to pre-existing immunity, determine whether the epitopes overlap with serotype-specific epitopes, and evaluate how overlapping motifs confer a broadly neutralizing phenotype to NAbs.

[0246] Depending on the method of exposure, the magnitude of the NAb response varies widely; individuals with natural immunity rarely have NAb titers exceeding 1:80 (humans) or 1:320 (macaques). In contrast, NAb titers exceeding 1:1000 can be easily achieved in response to delivery of modest doses of vector (Greig JA, et al. Vaccine. 2016;34(50):6323-29; Greig JA, et al. Hum Gene Ther Clin Dev. 2017;28(1):39-50; Greig JA, et al. PLoS One. 2014;9(11):e112268). In this study, mice receiving the highest vector dose resulting in the highest NAb titers had measurable variation in mutant vector binding, suggesting that the strength of the NAb response influences mutation efficiency. In most cases, studies aim to reduce an individual's NAb titer below the threshold that prevents gene transfer (1:10 for intravenous administration) (Chicoine (LG, et al. Mol Ther. 2014;22(2):338-47; Wang L, et al. Hum Gene Ther. 2011;22(11):1389-1401). Mutant capsids engineered based on a single neutralizing epitope that confers escape only to high-titer sera would not significantly increase the number of individuals eligible for AAV gene therapy, as lower titers would still be above the threshold at which transduction is significantly inhibited.

[0247] The minimal mutation required to reduce PAV9.1 binding at Q590 in HVR VIII, even after a conservative amino acid substitution to asparagine, conferred a liver-detargeting phenotype to the resulting mutant. Mutations in the HVR V portion of the epitope also reduced liver transduction. These results are consistent with previous observations that these residues in HVRs V and VIII play essential roles in liver transduction, as well as previous reports of mapping neutralizing AAV epitopes that overlap with regions essential for gene transfer (Adachi K, et al. Nature Communications. 2014;5:3075; Tseng TS, et al. J Virol. 2015;89(3):1794-8). 08). This suggests that engineering mutants that can evade NAb while maintaining the parental transduction profile is challenging. For some cardiac and muscular indications, where liver transduction may be less important, this change in tropism may be acceptable. Notably, the majority of mutants maintained WT.AAV9 levels of transduction in peripheral organs at both doses.

[0248] The RGNRQ mutation showed modest binding modifications in the presence of polyclonal antibodies, but exhibited an AAV2-like transduction profile and poor transduction of all peripheral organs, not just the liver. Collectively, these data demonstrate the importance of integrating knowledge of mapped neutralizing epitopes with available information on AAV functional domains. Generating capsids capable of evading NAb is not sufficient, as the capsid is only useful if it can still fulfill its primary function of target tissue transduction. Recent studies have used this strategy to incorporate multiple epitopes of AAV1, generating AAV1-based vectors capable of evading NAb while maintaining an AAV1-like transduction profile (Tse LV, et al. Proc Natl Acad Sci USA.2017;114(24),E4812-21).

[0249] Collectively, this study provides important information for the design of AAV9-based vectors that can evade humoral immune responses. Future studies are needed to inform the design of next-generation capsids and further understand the complexity of the NAb response to AAV9 vectors.

[0250] (Sequence listing free text) The following information is an identification number <223> Below we provide an array containing free text. [Table 25]

[0251] All documents cited herein are incorporated by reference, including U.S. Provisional Patent Applications Nos. 62 / 722,388 and 62 / 722,382, both filed August 24, 2018, U.S. Provisional Patent Applications Nos. 62 / 703,670 and 62 / 703,673, both filed July 26, 2018, U.S. Provisional Patent Applications Nos. 62 / 677,471 and 62 / 677,474, both filed May 29, 2018, U.S. Provisional Patent Application No. 62 / 667,585, filed May 29, 2018, and U.S. Provisional Patent Application No. 62 / 635,964, filed February 27, 2018. U.S. Provisional Patent Application No. 63 / 667,881, filed May 7, 2018; U.S. Provisional Patent Application No. 62 / 667,888, filed May 7, 2018; U.S. Provisional Patent Application No. 62 / 667,587, filed May 6, 2018; U.S. Provisional Patent Application No. 62 / 663,797, filed April 27, 2018; U.S. Provisional Patent Application No. 62 / 663,788, filed April 27, 2018; and U.S. Provisional Patent Application No. 62 / 635,968, filed February 27, 2018, are incorporated by reference. SEQ ID NOs. referenced herein and appearing in the accompanying Sequence Listing are incorporated by reference. While the present invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A composition comprising a mixed population of recombinant adeno-associated viruses (rAAVs), each of said rAAVs comprising: (a) an AAV capsid comprising about 60 capsid vp1, vp2, and vp3 proteins, wherein the vp1, vp2, and vp3 proteins are: a heterogeneous population of vp1 proteins produced from nucleic acid sequences encoding selected AAV vp1 amino acid sequences; a heterogeneous population of vp2 proteins produced from nucleic acid sequences encoding selected AAV vp2 amino acid sequences; a heterogeneous population of vp3 proteins produced from nucleic acid sequences encoding selected AAV vp3 amino acid sequences; an AAV capsid, wherein the vp1, vp2, and vp3 proteins comprise a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in asparagine-glycine pairs in the AAV capsid, and optionally further comprise a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change, with the proviso that the rAAV is not AAVhu68; (b) a vector genome in said AAV capsid, said vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence, and a non-AAV nucleic acid sequence encoding said product operably linked to a sequence that directs expression of the product in a host cell.

2. 2. The composition of claim 1, wherein the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid / isoaspartic acid pair, or a combination thereof.

3. 2. The composition of claim 1, wherein the capsid further comprises deamidated glutamine that is deamidated to (α)-glutamic acid, γ-glutamic acid, an interconverted (α)-glutamic acid / γ-glutamic acid pair, or a combination thereof.

4. The composition of any one of claims 1 to 4, wherein the capsid comprises 4 to 5 highly deamidated asparagines in asparagine-glycine pairs.

5. 6. The composition of any one of claims 1-5, wherein the capsid comprises 65% to 100% deamidated asparagine at position 57 relative to AAV8 or AAV9 numbering as determined using mass spectrometry.

6. (a) an rAAV having an AAV8 capsid, wherein the composition further comprises a subpopulation in which at least 70% to 100% of the N in the capsid are deamidated at positions N57, N263, N385, N514, and / or N540 of SEQ ID NO: 6 (encoding AAV8 vp1), based on the numbering of AAV8 vp1 with the first M; (b) a rAAV having an AAV9 capsid, further comprising a subpopulation in which at least 65% to 100% of the N in said capsid are deamidated at positions N57, N329, N452, and / or N512, based on the numbering of SEQ ID NO: 7 (encoding AAV9 vp1) with the first M; (c) AAVrhlO capsids (AAVrhlO vp1) further comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of the N's are deamidated at one or more N-G pairs at positions N263, N385, and / or N514, based on the numbering of SEQ ID NO: 112 (encoding AAVrhlO vp1) with the first M. 0), or 6. The composition of any of claims 1 to 5, comprising: (d) an rAAV having an AAVhu37 capsid (AAVhu37), further comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of N's are deamidated at one or more N-G pairs at positions N263, N385, and / or N514, based on the numbering of SEQ ID NO: 36 (encoding AAVhu37 vp1) with the first M.

7. The composition comprises: (a) an rAAV having an AAV1 capsid comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of the N's are deamidated at one or more N-G pairs at positions N57, N383, N512, and N718, based on the numbering of SEQ ID NO: 1, which is based on the numbering of the predicted vp1 amino acid sequence with the first M; (b) an rAAV having an AAV3B capsid comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of the N's are deamidated at one or more N-G pairs at positions N57, N382, N512, and N718, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 2; (c) an rAAV having an AAV5 capsid comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of the N's are deamidated at one or more N-G pairs at positions N56, N347, N347, N509, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 3; (d) an rAAV having an AAV7 capsid comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of the N's are deamidated at one or more N-G pairs at positions N41, N57, N384, and N514, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 4; (e) an rAAV having an AAVrh32.33 capsid comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of the N's are deamidated at one or more N-G pairs at positions N57, N264, N292, and N318, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO:5; or (f) a rAAV4 vector comprising a subpopulation of vp1, vp2, and / or vp3 in which at least 70% to 100% of N's are deamidated at one or more N-G pairs at positions N56, N264, N318, and N546, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO:

111.

8. 8. The composition of any of claims 1 to 7, wherein the capsid comprises 80% to 100% deamidated asparagine at position 57 relative to AAV8 or AAV9 numbering.

9. 9. The composition of any one of claims 1 to 8, wherein all or a subpopulation of the AAV vp1 and / or vp3 proteins have a truncation of about 1 to about 5 amino acids at their N-terminus.

10. 10. The composition of any one of claims 1 to 9, wherein all or a subset of the AAV vp1 and / or vp3 proteins have a truncation of about 1 to about 5 amino acids at their C-terminus.

11. 1. A method for reducing deamidation of AAV capsids, the method comprising producing AAV capsids from nucleic acid sequences comprising modified AAV vp codons, the nucleic acid sequences being independently modified at one to three of the asparagine-glycine pairs relative to a reference AAV vp1 sequence such that the modified codons encode amino acids other than glycine. The method includes a modified glycine codon.

12. 1. A method for reducing deamidation of AAV capsids, the method comprising producing AAV capsids from a nucleic acid sequence comprising modified AAV vp codons, the nucleic acid sequence comprising asparagine codons independently modified with at least one asparagine-glycine pair relative to a reference AAV vp1 sequence, such that the modified codon encodes an amino acid other than asparagine.

13. 1. A method for increasing the titer, efficacy, or transduction of a recombinant AAV, the method comprising producing an AAV capsid from a nucleic acid sequence comprising at least one AAV vp codon modified to change the asparagine or glycine of at least one asparagine-glycine pair in the capsid to a different amino acid.

14. The method of any one of claims 11 to 13, wherein the modified codon is within the v2 and / or vp3 region.

15. The method of any one of claims 11 to 13, wherein the asparagine-glycine pair in the vp1 unique region is retained in the modified rAAV.

16. The deamidation site is (a) for the AAV8 capsid, N57, N263, N385, N514, and / or N540 of SEQ ID NO: 6 (encoding AAV8 vp1), based on the numbering of AAV8 vp1 with the first M; (b) for the AAV9 capsid, N57, N329, N452, and / or N512, based on the numbering of SEQ ID NO: 7 (encoding AAV9 vp1) with the first M; (c) for an AAVrhlO capsid, N57, N263, N385, and / or N514, based on the numbering of SEQ ID NO: 112 (encoding AAVrhlO vp1) with the first M; or (d) the AAVhu37 capsid is modified at a position other than N57, N263, N385, and / or N514, based on the numbering of SEQ ID NO: 36 (encoding AAVhu37 vp1) with the first M.

17. 17. The method of claim 16, wherein the modified deamidation site is selected from a site in Table F, Table G, or Table H.

18. The deamidation site is (a) for the AAV1 capsid, N57, N383, N512, and / or N718, based on the numbering of SEQ ID NO: 1, which is based on the numbering of the predicted vp1 amino acid sequence with the first M; (b) for an AAV3B capsid, N57, N382, N512, and / or N718, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO:2; (c) for an AAV5 capsid, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 3, N56, N347, N347, and / or N509; (d) for an AAV7 capsid, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 4, N41, N57, N384, and / or N514; (e) for an AAVrh32.33 capsid, N57, N264, N292, and / or N318, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 5; or (f) the AAV4 capsid is modified at positions other than N56, N264, N318, and / or N546, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO:

111.

19. 19. The method of claim 18, wherein the modified deamidation site is selected from a site in Table A, Table B, Table C, Table D, Table E, Table F, Table G, or Table H.

20. The method of any one of claims 11 to 19, wherein each modified codon encodes a different amino acid.

21. The method of any one of claims 11 to 19, wherein two or more modified codons encode the same amino acid.

22. A mutant rAAV comprising an AAV capsid with reduced deamidation compared to an unmodified AAV capsid, produced using the method of any one of claims 11 to 21.

23. Based on VP1 numbering, (a) AAV8 G264A / G541A (SEQ ID NO: 23); (b) AAV8 G264A / G541A / N499Q (SEQ ID NO: 115); (c) AAV8 G264A / G541A / N459Q (SEQ ID NO: 116); (d) AAV8 G264A / G541A / N305Q / N459Q (SEQ ID NO: 117); (e) AAV8 G264A / G541A / N305Q / N499Q (SEQ ID NO: 118); (f) AAV8 G264A / G541A / N459Q / N499Q (SEQ ID NO: 119); (g) AAV8 G264A / G541A / N305Q / N459Q / N499Q (SEQ ID NO: 120); (h) AAV8 G264A / G515A (SEQ ID NO: 21); (i) AAV8G515A / G541A (SEQ ID NO: 25); (j) AAV8 G264A / G515A / G541A (SEQ ID NO: 27); (k) AAV9 G330 / G453A (SEQ ID NO: 29); (l) AAV9G330A / G513A (SEQ ID NO: 31); (m) AAV9G453A / G513A (SEQ ID NO: 33), and / or (n) The mutant rAAV of claim 22, having a mutant AAV capsid having a capsid protein with one or more of the following substitutions: G330 / G453A / G513A (SEQ ID NO: 35).

24. 23. The mutant rAAV of claim 22, having a mutant AAV capsid having a capsid protein with one or more of the following substitutions based on the AAV8 VP1 numbering: N263A, N514A, or AAV N540A.

25. 23. The mutant rAAV of claim 22, having a mutant AAV capsid having a capsid protein in which the wild-type NG pair is retained at positions N57, N94, N263, N305, G386, Q467, N479, and / or N653.

26. 1. A composition comprising a population of rAAV having increased titer, efficacy, or transduction, wherein the composition comprises a rAAV having a capsid modified to have reduced total deamidation compared to a rAAV having a deamidation pattern according to any one of Table A (AAV1), Table B (AAV3B), Table C (AAV5), Table D (AAV7), Table E (AAVrh32.33), Table F (AAV8), Table G (AAV9), or Table H (AAVhu37), with the proviso that the rAAV is not AAVhu68.

27. The rAAV (a) for the AAV8 capsid, N57, N263, N385, N514, and / or N540 of SEQ ID NO: 6 (encoding AAV8 vp1), based on the numbering of AAV8 vp1 with the first M; (b) for the AAV9 capsid, N57, N329, N452, and / or N512, based on the numbering of SEQ ID NO: 7 (encoding AAV9 vp1) with the first M; (c) for an AAVrhlO capsid, N57, N263, N385, and / or N514, based on the numbering of SEQ ID NO: 112 (encoding AAVrhlO vp1) with the first M; or (d) for an AAVhu37 capsid, having a modified deamidation site at a position other than N57, N263, N385, and / or N514, based on the numbering of SEQ ID NO: 36 (encoding AAVhu37 vp1) with the first M.

28. The rAAV (a) for the AAV1 capsid, N57, N383, N512, and / or N718, based on the numbering of SEQ ID NO: 1, which is based on the numbering of the predicted vp1 amino acid sequence with the first M; (b) for an AAV3B capsid, N57, N382, N512, and / or N718, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO:2; (c) for an AAV5 capsid, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 3, N56, N347, N347, and / or N509; (d) for an AAV7 capsid, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 4, N41, N57, N384, and / or N514; (e) for an AAVrh32.33 capsid, N57, N264, N292, and / or N318, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 5; or (f) for an AAV4 capsid, the composition of claim 26 has a modified amino acid sequence deamidation site modified at a position other than N56, N264, N318, and / or N546, based on the numbering of the predicted vp1 amino acid sequence with the first M, with reference to the numbering of SEQ ID NO: 111.

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