Novel adeno-associated virus (AAV) vectors, AAV vectors with reduced capsid deamidation, and their use

Abstract

To provide compositions comprising AAV-based constructs for delivery of heterologous molecules which have stable receptor binding and / or stable capsid, avoid neutralizing antibodies and / or retain purity during storage.SOLUTION: A recombinant adeno-associated virus (rAAV) vector comprises an AAV capsid having a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 protein and a heterogeneous population of vp3 proteins. The capsid contains modified amino acids as compared to the encoded VP1 amino acid sequence, the capsid containing highly deamidated asparagine residues at asparagine-glycine pair, and further comprising multiple other, less deamidated asparagine and optionally glutamine residues.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Description of Federally Sponsored Research This invention was made with government support under P01HL059407 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Reference to a Sequence Listing Submitted via EFS-WEB The contents of the filed text of the sequence listing named "18-8592PCT_Sequence_Listing_ST25", created on February 27, 2019, and electronically submitted via EFS-Web with this application, are hereby incorporated by reference in their entirety into this specification.

Background Art

[0003] The adeno-associated virus (AAV) capsid has an icosahedral structure and consists 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 (~535aa) is contained within the C-terminuses of both VP1 and VP2, and the shared VP3 sequence is primarily involved in the overall capsid structure. Due to the structural flexibility of the VP1 / VP2 intrinsic region, as well as the lower expression of VP1 and VP2 monomers compared to VP3 monomers in the assembled capsid, VP3 is the only capsid protein 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 heavily 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). While the structures of several serotypes have been published (protein databank (PDB) numbers 1LP3, 4RSO, 4V86, 3UX1, 3KIC, 2QA0, and 2G8G from the Research Collaboratory for Structural Bioinformatics (RCSB) database for the structural entries of AAV2, AAVrh.8, AAV6, AAV9, AAV3B, AAV8, and AAV4, respectively), there is very little information in the literature regarding surface modifications of these capsids. Studies suggest that intracellular phosphorylation of the capsid occurs at specific tyrosine residues (Zhong L, et al. Virology. 2008;381(2):194-202).Despite the prediction of glycosylation sites in major VP3 sequences, 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), other AAV serotypes have not yet been evaluated for capsid glycosylation.

[0004] AAV gene therapy vectors typically undergo little molecular scrutiny in the development and manufacturing of recombinant protein therapies. Post-translational modifications (PTMs) of AAV capsids are largely unexplored, so little is known about their potential impact on function or strategies for controlling PTM levels in manufactured AAV therapies.

[0005] The diversity of post-translational modifications in non-gene therapies and protein therapies complicates their drug development. (Jenkins, N, Murphy, L, and Tyther, R (2008). Post-translational modifications of recombinant proteins: significance for 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 anthrax vaccines. (Powell BS, et al. Proteins. 2007;68(2):458-79; 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. The purpose of spontaneous deamidation is not fully understood, but previous studies have suggested that this event indicates the relative age of a protein and plays a role in a molecular clock for regulating its turnover (Robinson NE and Robinson AB. Proc Natl Acad Sci USA. 2001;98(3):944-9).

[0006] Deamidation occurs when the amide group of asparagine, or less frequently, glutamine, is nucleophilically attacked by an adjacent nitrogen atom, resulting in the loss of the amide group. This process yields a succinimidyl intermediate (Yang H and Zubarev RA. Electrophoresis. 2010;31(11):1764-72), which is broken down 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). Short synthetic peptide studies suggest that this hydrolysis yields a 3:1 mixture of isoaspartic acid to aspartic acid (Geiger T. and Clarke SJ Biol Chem. 1987;262(2):785-94).

[0007] There remains a need for compositions containing AAV system constructs for delivering heterologous molecules that have stable receptor binding and / or stable capsids, avoid neutralizing antibodies, and / or maintain purity during storage. [Overview of the project]

[0008] In one embodiment, a composition comprising a mixed population of recombinant adeno-associated viruses (rAAV), each of which is an AAV capsid comprising about 60 capsid vp1 proteins, vp2 proteins, and vp3 proteins, wherein the vp1, vp2, and vp3 proteins are heterogeneous populations of vp1 proteins produced from nucleic acid sequences encoding selected AAV vp1 amino acid sequences, heterogeneous populations of vp2 proteins produced from nucleic acid sequences encoding selected AAV vp2 amino acid sequences, and heterogeneous populations of vp3 proteins produced from nucleic acid sequences encoding selected AAV vp3 amino acid sequences, wherein the vp1, vp2, and vp3 proteins have amino acid modifications comprising at least two highly deamidated asparagine (N) in the asparagine-glycine pair in the AAV capsid. A composition is provided, comprising (b) an AAV capsid comprising a subpopulation of AAV amino acids, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein 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 reverse terminal repeat sequence, and a non-AAV nucleic acid sequence encoding a product that is operably linked to a sequence that directs the expression of the product in a host cell.

[0009] In certain embodiments, deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, interconverted aspartic acid / isoaspartic acid pairs, or combinations thereof. In certain embodiments, the capsid further comprises deamidated glutamine(s) that is deamidated to (α)-glutamic acid, γ-glutamic acid, interconverted (α)-glutamic acid / γ-glutamic acid pairs, or combinations thereof.

[0010] In a further embodiment, recombinant adeno-associated virus (rAAV) is an AAVrh79 capsid, and (1) a heterogeneous population of AAVrh79 vp1 proteins selected from vp1 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-738 of SEQ ID NO: 2, vp1 proteins produced from SEQ ID NO: 1, or vp1 proteins produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 1 that encodes the predicted amino acid sequence of 1-738 of SEQ ID NO: 2; vp2 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 138-738 of SEQ ID NO: 2, vp2 proteins produced from a sequence containing at least nucleotides 412-2214 of SEQ ID NO: 1, or vp2 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO: 1 that encodes the predicted amino acid sequence of at least approximately 138-738 of SEQ ID NO: 2. AAVrh79 is selected from a heterogeneous population of vp2 proteins, vp3 proteins produced by expression from a nucleic acid sequence encoding at least approximately 204-738 of the predicted amino acid sequence of SEQ ID NO: 2, vp3 proteins produced from a sequence containing at least nucleotides 610-2214 of SEQ ID NO: 1, or vp3 proteins produced from a nucleic acid sequence encoding at least approximately 204-738 of the predicted amino acid sequence of SEQ ID NO: 2, which is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO: 1.A heterogeneous population of vp3 protein, including the AAVrh79 capsid protein and / or (2) a heterogeneous population of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, a heterogeneous population of vp2 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 at least approximately amino acids 138-738, and a heterogeneous population of vp3 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 at least 2 A recombinant adeno-associated virus is provided, comprising (B) an AAVrh79 capsid comprising one or more heterogeneous populations having amino acid modifications including one highly deamidated asparagine (N), optionally further comprising a subpopulation having other deamidated amino acids, wherein deamidation results in an amino acid change; and (B) a vector genome in the AAVrh79 capsid, wherein the vector genome comprises a nucleic acid molecule having an AAV reverse terminal repeat sequence, and a non-AAV nucleic acid sequence encoding a product that is operably linked to a sequence that directs the expression of the product in a host cell.

[0011] In another embodiment, a method for transducing target tissue is provided. In one embodiment, the method comprises administering an AAV having the AAVrh79 capsid described herein. In one embodiment, a method for transducing liver tissue is provided, comprising administering an AAV having the AAVrh79 capsid. In another embodiment, a method for transducing muscle tissue is provided, comprising administering an AAV having the AAVrh79 capsid.

[0012] In yet another embodiment, a method for reducing the deamidation of the AAVrh79 capsid is provided. In one embodiment, the method comprises producing an AAVrh79 capsid from a nucleic acid sequence containing a modified AAVrh79 VP codon, wherein the nucleic acid sequence comprises a glycine codon independently modified with 1 to 4 asparagine-glycine pairs located at positions N57, N263, N385, and / or N514 of SEQ ID NO: 2, such that the modified codon encodes an amino acid other than glycine. In another embodiment, the method comprises producing an AAVrh79 capsid from a nucleic acid sequence containing a modified AAVrh79 vp codon, wherein the nucleic acid sequence comprises a glycine codon independently modified with 1 to 4 asparagine-glycine pairs located at positions N94, N254, N305, N410, and / or N479 of SEQ ID NO: 2.

[0013] In further embodiments, rAAV8.AR2.08 is (A) an AAV8.AR2.08 capsid, (1) a heterogeneous population of AAV8.AR2.08vp1 proteins selected from (1) a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 18 from 1 to 738, a vp1 protein produced from SEQ ID NO: 17, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 17 encoding the predicted amino acid sequence of SEQ ID NO: 18 from 1 to 738, an AAV8.AR2.08vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately amino acids 138 to 738 of SEQ ID NO: 18, a vp2 protein produced from a sequence containing at least nucleotides 411 to 2214 of SEQ ID NO: 17, or at least approximately AAV8.2 includes a heterogeneous population of AAV8.AR2.08vp2 proteins selected from vp2 proteins produced from nucleic acid sequences that are at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO: 17, encoding the predicted amino acid sequence of amino acids 138-738; vp3 proteins produced by expression from nucleic acid sequences that are at least approximately 204-738 of the predicted amino acid sequence of SEQ ID NO: 18; vp3 proteins produced from sequences containing at least nucleotides 607-2214 of SEQ ID NO: 17; or vp3 proteins produced from nucleic acid sequences that are at least 70% identical to at least nucleotides 607-2214 of SEQ ID NO: 17, encoding the predicted amino acid sequence of at least approximately 204-738 of SEQ ID NO: 18.(1) a capsid protein, and / or (2) a heterogeneous population of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, a heterogeneous population of vp2 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 at least approximately amino acids 138-738, and a heterogeneous population of vp3 protein which is the product of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO: 18, wherein the vp1, vp2, and vp3 proteins are at least two highly deamidated asparagine in the asparagine-glycine pair of SEQ ID NO: 18 (N) AAV8.AR2.08 is provided, comprising an AAV8.AR2.08 capsid containing one or more heterogeneous populations, each containing a subpopulation having amino acid modifications including (N), and optionally further containing other deamidated amino acids, wherein deamidation results in an amino acid change; and (B) a vector genome in the AAV8.AR2.08 capsid, wherein the vector genome contains a nucleic acid molecule containing an AAV reverse-terminal repeat sequence, and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs the expression of the product in a host cell.

[0014] In another embodiment, a method for transducing target tissue is provided. In one embodiment, the method comprises administering an AAV having the AAV8.AR2.08 capsid described herein. In one embodiment, a method for transducing liver tissue is provided, comprising administering an AAV having the AAV8.AR2.08 capsid. In another embodiment, a method for transducing muscle tissue is provided, comprising administering an AAV having the AAV8.AR2.08 capsid.

[0015] In yet another embodiment, a method for reducing the deamidation of the AAV8.AR2.08 capsid is provided. In one embodiment, the method comprises producing an AAV8.AR2.08 capsid from a nucleic acid sequence containing a modified AAV8.AR2.08 vp codon, the nucleic acid sequence containing a glycine codon independently modified with 1 to 4 of the asparagine-glycine pairs located at positions N57, N263, N385, N514, and / or N540 of SEQ ID NO: 18, such that the modified codon encodes an amino acid other than glycine. In another embodiment, the method comprises producing an AAV8.AR2.08 capsid from a nucleic acid sequence containing a modified AAV8.AR2.08 vp codon, wherein the nucleic acid sequence contains a glycine codon independently modified by one to four of the asparagine-glycine pairs located at positions N94, N254, N305, N521, N590, Q601, N653, and / or N665 of SEQ ID NO: 18.

[0016] In a particular embodiment, rAAV5.5.9 is an AAV5.5.9 capsid, and (1) a heterogeneous population of AAV5.5.9 vp1 proteins selected from (A) a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 10 from 1 to 736, a vp1 protein produced from SEQ ID NO: 9, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 9, which encodes the predicted amino acid sequence of SEQ ID NO: 1 from 1 to 736; an AAV5.5.9 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 10 from at least approximately 138 to 736, a vp2 protein produced from a sequence containing at least nucleotides 412 to 2211 of SEQ ID NO: 9, or an AAV5.5.9 vp2 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 10 from at least approximately 138 to 736, which is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 9. AAV5.5.9 vp3 protein is selected from a heterogeneous population of vp2 proteins, vp3 protein produced by expression from a nucleic acid sequence encoding at least approximately 203-736 of the predicted amino acid sequence of SEQ ID NO: 10, vp3 protein produced from a sequence containing at least nucleotides 607-2211 of SEQ ID NO: 9, or vp3 protein produced from a nucleic acid sequence encoding at least approximately 203-736 of the predicted amino acid sequence of SEQ ID NO: 10, which is at least 70% identical to at least nucleotides 607-2211 of SEQ ID NO: 9.9 A heterogeneous population of vp3 protein, including the AAVG5 capsid protein and / or (2) a heterogeneous population of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10, a heterogeneous population of vp2 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10 at least approximately amino acids 138-736, and a heterogeneous population of vp3 protein which is the product of a nucleic acid sequence encoding at least amino acids 203-726 of SEQ ID NO: 10, wherein the vp1, vp2, and vp3 proteins are at least 2 in the asparagine-glycine pair of SEQ ID NO: 10 AAV5.5.9 is provided, comprising (B) a vector genome in the AAV5.5.9 capsid, the vector genome comprising a nucleic acid molecule containing an AAV reverse-terminal repeat sequence, and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs the expression of the product in a host cell.

[0017] In another embodiment, a method for transducing target tissue is provided. In one embodiment, the method comprises administering an AAV having the AAV5.5.9 capsid described herein. In one embodiment, a method for transducing liver tissue is provided, comprising administering an AAV having the AAV5.5.9 capsid. In another embodiment, a method for transducing muscle tissue is provided, comprising administering an AAV having the AAV5.5.9 capsid.

[0018] In yet another embodiment, a method for reducing the deamidation of the AAV5.5.9 capsid is provided. In one embodiment, the method comprises producing an AAV5.5.9 capsid from a nucleic acid sequence containing a modified AAV5.5.9 vp codon, the nucleic acid sequence containing a glycine codon independently modified with 1 to 4 of the asparagine-glycine pairs located at positions N57, N319, N442, and / or N502 of SEQ ID NO: 10, such that the modified codon encodes an amino acid other than glycine. In another embodiment, the method comprises producing an AAV5.5.9 capsid from a nucleic acid sequence containing a modified AAV5.5.9 vp codon, wherein the nucleic acid sequence contains a glycine codon independently modified by 1 to 4 of the asparagine-glycine pairs located at positions N35, N113, N204, N217, N243, N249, N293 / 294, N304, N399 / 400, N505, Q589, N618, N641, N653, N658, and / or N699 of SEQ ID NO: 10.

[0019] In another embodiment, a composition is provided comprising a mixed population of recombinant AAVrh79, AAV8.AR2.08, or AAV5.5.9 as described herein.

[0020] In yet another embodiment, recombinant AAVs (rAAVs) described herein are provided for delivering a desired gene product to an object that requires it.

[0021] In another embodiment, an rAAV production system useful for producing the rAAV described herein is provided. In one embodiment, the system includes (a) an AAVrh79, AAV8.AR2.08, or AAV5.5.9 capsid nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2, 10, or 18; (b) a nucleic acid molecule suitable for packaging into an AAV capsid, wherein the nucleic acid molecule comprises a non-AAV nucleic acid sequence encoding a gene product operably linked to at least one AAV reverse terminal repeat (ITR) and a sequence that directs the expression of the product in a host cell; and (c) sufficient AAV rep and helper functions to enable packaging the nucleic acid molecule into a recombinant AAV capsid.

[0022] These and other aspects of the present invention will become apparent from the following detailed description of the invention. [Brief explanation of the drawing]

[0023] [Figure 1A]Figures 1A–1G show electrophoretic analysis of AAV8 VP isoforms. (Figure 1A) The figure shows the mechanism by which an asparagine residue is nucleophilically attacked by an adjacent nitrogen atom to form a succinimidyl intermediate. This intermediate is then hydrolyzed and broken down into a mixture of aspartic acid and isoaspartic acid. The beta carbon is labeled in this way. The figure was generated with BIOVIA Draw 2018. (Figure 1B) 1 μg of AAV8 vector was run on denatured 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. The box regions are a=VP1, b=VP2, c=VP3, and d=internal tropomyosin markers (arrows: tropomyosin spots with MW=33kDa and pI=5.2). Isoelectric focusing was performed in the pI range of 4–8. Figures 1E–1G) Results of isoelectric focusing performed in the pI range of 4–8. 1e11 GC wtAAV8 (Figure 1E) or mutant (Figures 1F and 1G) vectors were analyzed by 2D gel electrophoresis and stained with Sypro Ruby. Protein labeling: A=VP1, B=VP2, C=VP3, D=chicken egg white con albumin marker, E=turbonuclease marker. Isoelectric focusing was performed in the pI range of 6–10. The main VP1 / 2 / 3 isoform spots are enclosed in circles, and the migration distance of the main spots on the marker is indicated by vertical lines (turbonuclease = dashed line, conalbumin = solid). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 2A]Figures 2A–2E show the analysis of asparagine and glutamine deamidation in the AAV8 capsid protein. (Figures 2A–2B) Electrospray ionization (ESI) mass spectrometry, as well as theoretical and observed masses, are shown for 3+ peptides (93–103) containing Asn-94 (Figure 2A) and Asp-94 (Figure 2B). (Figures 2C–2D) ESI mass spectrometry, as well as theoretical and observed masses, are shown for 3+ peptides (247–259) containing Asn-254 (Figure 2C) and Asp-254 (Figure 2D). The observed mass shifts for Asn-94 and Asn-254 were 0.982 Da and 0.986 Da, respectively, against a theoretical mass shift of 0.984 Da. (Figure 2E) The deamidation rates at specific asparagine and glutamine residues of interest are shown for AAV8 trypsin peptides purified by different methods. Bars indicating deamidation at asparagine residues containing N+1 glycine are shaded. This includes residues determined to be at least 2% deamidated in at least one of the analyzed preparations. Data are expressed as mean ± standard deviation. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 3A]Figures 3A to 3E show structural modeling and deamidation site analysis of the AAV8 VP3 monomer. (Figure 3A) The AAV8 VP3 monomer (PDB identifier: 3RA8) is shown in coil representation. The ribbon color indicates the degree of relative flexibility (blue = stiffest / normal temperature coefficient, red = most flexible / high temperature coefficient). The spheres represent the target residue. The magnified view shows ball and stick representations of the target residue and surrounding residues, showing the local protein structure (blue = nitrogen, red = oxygen). Underlined residues are NG motifs. Figures 3B to 3E: Show isoasparagine system models of deamidated asparagine with N+1 glycine. The 2FoFc electron density map (1 sigma level) is generated from the refinement of the AAV8 crystal structure (PDB number: 3RA8) using the asparagine model (Figure 3B)N410, compared with the isoaspartic acid models (Figure 3C)N263, (Figure 3D)N514, and (Figure 3E)N540. The electron density map is shown in magenta grid. Beta carbons are labeled in this manner. Arrows indicate the electron density corresponding to the R group of the residue of interest. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 4A] Figures 4A–4D show the determination of factors influencing AAV8 capsid deamidation. AAV8 preparations were incubated at 70°C for 3 or 7 days (Figure 4A), exposed to pH 2 or pH 10 for 7 days (Figure 4B), or prepared for mass spectrometry using D2O instead of H2O (Figure 4C) to identify possible sources of deamidation not specific to AAV capsid formation. (Figure 4D) Dot blots of vectors treated as in Figure 4A, using B1 antibody (reacting on denatured capsids) and AAV8 structure-specific antibody (reacting on intact capsids) to assess the structural integrity of the capsids. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 5A]Figures 5A and 5B show the deamidation frequencies of non-AAV proteins. Deamidation rates are shown for two non-AAV recombinant proteins containing NG motifs that are likely to be deamidated: human carbon dehydrase (Figure 5A) and rat phenylalanine hydroxylase (Figure 5B), for comparison with AAV deamidation rates. [Figure 5B] Same as above. [Figure 6] Figure 6 compares the AAV8 percent amidification calculated using data analysis pipelines from two institutions. The deamidation rates at specific asparagine and glutamine residues of interest are shown for the AAV8 trypsin peptide evaluated by two different institutions. [Figure 7A] Figures 7A–7C illustrate functional asparagine substitution at non-NG sites with high lot-to-lot variability. (Figure 7A) Titer of wtAAV8 and mutant vectors produced by small-scale triple transfection in 293 cells, measured by quantitative PCR (qPCR). Titer is reported relative to the wtAAV8 control. Transduction efficiency was measured as shown in Figure 8B. Titer and transduction efficiency are normalized to the wtAAV8 control values. (Figure 7B) Representative luciferase images at 14 days post-injection for mice receiving wtAAV8.CB7.ffluc and N499Q capsid mutant vectors. (Figure 7C) Luciferase expression at 14 days post-injection from C57BL / 6 mice intravenously injected with wtAAV8 or mutant vectors (n=3 or 4), measured by luciferase imaging and reported in total flow units, at 14 days of the study period. 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 in vitro analysis of the effect of gene deamidation on vector performance. (Figure 8A) Titer of wtAAV8 and gene deamidated mutant vectors produced by small-scale triple transfection in 293 cells, measured by quantitative PCR (qPCR). Titer is reported relative to the wtAAV8 control. Deamidated NG sites (patterned bars), deamidated sites (white bars), and highly variable sites (black bars) are shown for wtAAV8 and the negative control. (Figure 8B) Transduction efficiency of the firefly luciferase-producing mutant AAV8 vector reported compared to the wtAAV8 control. Transduction efficiency is measured by luminescence units generated for each GC added to HUH7 cells and determined by transduction using crude vectors at multiple dilutions. Transduction efficiency data are normalized to a reference. All data are expressed as mean ± standard deviation. [Figure 8B] Same as above. [Figure 9A] Figures 9A–9D illustrate the correlation between time-dependent vector activity loss and progressive deamidation. (Figure 9A) Vector production (DNAseI-resistant genomic copy, GC) over time in triple-transfected HEK293 cells producing an AAV8 vector packaging a luciferase reporter gene. GC levels are normalized to the maximum observed value. (Figure 9B) Huh7 cells were transduced using purified time-dependent vectors. Transduction efficiency (luminescence units per GC added to target cells) was measured using multiple dilutions of purified time-dependent vector samples, as shown in Figure 8B. Error bars represent the standard deviation of at least 10 technical replications for each sample time. Deamidation of the AAV8 NG site (Figure 9C) and non-NG site (Figure 9D) of the vector collected 1, 2, and 5 days after transfection. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A]Figures 10A–10D illustrate the effect of asparagine stabilization on vector performance. Figure 10A shows the titers of wtAAV8 and +1 positional mutant vectors produced by small-scale triple transfection in 293 cells, measured by quantitative PCR (qPCR). Titers are reported relative to the wtAAV8 control. Figure 10B shows the transduction efficiency of the firefly luciferase-producing mutant AAV8 vector compared to the wtAAV8 control. Transduction efficiency was measured using crude vector material as shown in Figure 8B. A two-sample t-test (*p<0.005) was performed to determine the significance of the difference between wtAAV8 and mutant transduction efficiency for G264A / G515A and G264A / G541A. Figure 10C shows luciferase expression at day 14 of the study period in the liver region of C57BL / 6 mice intravenously injected with wtAAV8 or mutant vectors (n=3-5), measured by luciferase imaging and reported in total flow units. Figure 10D shows the titer and transduction efficiency of multi-site AAV8 mutant vectors producing firefly luciferase, compared to the wtAAV8 control. All data are expressed as mean ± standard deviation. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 11A]Figures 11A–11C show the analysis of asparagine and glutamine deamidation in the AAV9 capsid protein. (Figure 11A) 1e11 GC wtAAV9 was analyzed by 2D gel electrophoresis and stained with Sypro Ruby. Protein labeling: 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. (Figure 11B) The deamidation rates at specific asparagine and glutamine residues of interest are shown for AAV9 trypsin 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 of the analyzed preparations were included. Data are expressed as mean ± standard deviation. (Figure 11C) The isoasparagine model of N512 is a 2FoFc electron density map (PDB number: 3UX1) generated by non-biased purification of the AAV9 crystal structure. The arrows indicate the electron density corresponding to the R group of residue N512. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Figures 11D–11F show the determination of factors influencing 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 expressed as mean ± standard deviation. (Figure 11F) Dot blot of vectors treated as in Figure 11D using B1 antibody (which reacts with denatured capsids) to assess the integrity of the capsid structure. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G]Figures 11G and 11H illustrate an in vitro analysis of the effect of gene deamidation on the vector performance of AAV9. (Figure 11G) Titer of wtAAV9 and gene deamidated mutant vectors produced by small-scale triple transfection in 293 cells, measured by quantitative PCR (qPCR). Titers are reported relative to the wtAAV9 control. Deamidated NG sites (patterned bars), deamidated sites (white bars), and highly variable sites (black bars) are shown for wtAAV8 and the negative control. (Figure 11H) Transduction efficiency of the mutant AAV9 vector producing firefly luciferase is reported compared to the wtAAV9 control. All data are expressed as mean ± standard deviation. [Figure 11H] Same as above. [Figure 11I] Figures 11I–11K show the in vitro efficacy of the AAV9 vector over time. (Figure 11I) Vector production (DNAseI-resistant genomic copy, GC) over time in triple-transfected HEK293 cells producing the AAV9 vector that packages the luciferase reporter gene. GC levels are normalized to the maximum observed value. (Figure 11J) Huh7 cells were transduced using the crude time-course vector. (Figure 11K) Transduction efficiency of the vector collected 1 day after transfection versus 5 days after 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 11J] Same as above. [Figure 11K] Same as above. [Figure 12A] Figure 12A provides the amino acid sequence alignments of AAV5.5.9 [SEQ ID NO: 10] (sometimes also called AAVG5), AAV9 [SEQ ID NO: 4], and AAVPHP.B [SEQ ID NO: 12], prepared using Clustal Omega 1.2.2 and its default parameters for alignment. [Figure 12B]Figures 12B–12E provide the nucleotide sequence alignments for AAV5.5.9[SEQ ID NO: 9], PHP.B[SEQ ID NO: 11], AAV9[SEQ ID NO: 3], and AAVhu68[SEQ ID NO: 14]. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 13A] Figure 13A provides amino acid sequence alignments of AAV8 triple mutant (AAV8T) [SEQ ID NO: 16], AAV8.AR2.08 [SEQ ID NO: 18] (sometimes also called AAVG3, AR2, or AAV.AR2), and AAV8 [SEQ ID NO: 20], prepared using Clustal Omega 1.2.2 and its default parameters for alignment. [Figure 13B] Figures 13B–13D provide nucleotide sequence arrangements for the AAV8 triple mutation [SEQ ID NO: 15], AAV8.AR2.08 [SEQ ID NO: 17], and AAV8 [SEQ ID NO: 19]. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 14A] Figure 14A shows the amino acid sequence alignment of AAVrh79[SEQ ID NO: 2] (sometimes called AAVG2), AAVrh10[SEQ ID NO: 24], and AAVhu37[SEQ ID NO: 22] using Clustal Omega 1.2.2 and its default parameters for alignment. [Figure 14B] Figures 14B–14D provide the nucleotide sequence arrangements for AAVrh79 [SEQ ID NO: 1], AAVrh10 [SEQ ID NO: 22], and AAVhu37 [SEQ ID NO: 21]. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 15A] Figures 15A and 15B illustrate the production yields of AAV8 triple, AAVhu68, AAV9, and AAVrh79 under small-scale and large-scale adjustments of the reference vector. [Figure 15B] Same as above. [Figure 16] Figure 16 shows the production purity of the large-scale adjustment in Figure 15B. [Figure 17A] Figures 17A-17D show the expression of luciferase in the liver and muscle tissue of male C57BL / 6 mice (n=5 / group) after intramuscular (IM) administration of 3 x 10¹¹ GC / mouse into the gastrocnemius muscle using a vector expressing firefly luciferase. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Figure 17E shows a comparison of the expression of AVV8 triple, AAVhu68, AAV9, AAV8, and AAVrh79 vectors after intramuscular administration of 10-13 GC / kg AAVrh79 to male and female cynomolgus macaques. [Figure 17F] Figure 17F shows the results of intramuscular administration of a vector expressing the secretory transgene (201Ig IA) into the gastrocnemius muscle of male RAG KO mice (n=5 / group) (3×10¹⁰ or 3×10¹¹ GC / mouse). [Figure 18A] Figure 18A shows the plasmid used in the barcode experiment of Example 5. [Figure 18B] Figure 18B shows the amounts of each AAV barcode variant injected into six black mice. The animals were sacrificed, tissue samples were taken, and DNA was isolated from each. Figure 18C shows the total vector distribution from three animals. Figure 18D shows the actual versus theoretical frequencies of the injected vector mixture. [Figure 18C] Same as above. [Figure 18D] Same as above. [Figure 19A] Figures 19A to 20C show the results of the barcode biodistribution experiment in Example 5. Individual tissue samples were analyzed for the frequency of individual barcodes in the sample-to-injection mixture for genome and cDNA. The results are shown for muscle (Figures 19A, 19B), heart (Figures 19C and 19D), and liver (Figures 19E and 19F). The magnification changes compared to theoretical frequencies are shown in Figures 20A to 20C. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above. [Figure 19E] Same as above. [Figure 19F] Same as above. [Figure 20A] Same as above. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 21] Figures 21 and 22 show the biodistribution of AAV8.AR2.08 in mice compared to AAV8. The results indicate that AAV8.AR2.08 is more liver-specific than AAV8. [Figure 22] Same as above. [Figure 23] Figure 23 compares AAV8, AAV8.AR2.08, and AAVrh79 in terms of potency and yield related to manufacturability. [Figure 24] Figure 24 shows the in vivo distribution of AAV8.AR2.08 in tissues (leftmost bar) compared to AAV8 (center and right bars). [Figure 25] Figures 25–28 show the results after administration of AAV vectors to non-human primates. Figure 25 provides details of the vectors and animals used in the study. Figure 26 quantifies the levels of GC and GFP detected in the livers of animals treated with AAV8, AAVrh79, or AAV8.AR2.08 vectors. Figure 27 summarizes the GFP expression levels in HNP livers. Figure 28 shows the levels of vectors detected in HNP-derived tissues treated with AAV8, AAVrh79, or AAV8.AR2.08 vectors. [Figure 26] Same as above. [Figure 27A] Same as above. [Figure 27B] Same as above. [Figure 28] Same as above. [Figure 29] Figure 29 shows the biological distribution of the AAVrh79 vector detected in various tissues. [Modes for carrying out the invention]

[0024] 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 recombinant AAVs, as well as compositions comprising them. Provided herein are novel rAAVs, as well as methods for reducing deamidation, and optionally other capsid monomer modifications. Further provided herein are modified rAAVs with reduced modification, which are useful in providing rAAVs having capsids that retain higher stability, potency, and / or purity.

[0025] "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle comprising 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 refers to "rAAV vector". The phrase "rAAV" can be used interchangeably with "AAV vector". rAAV is a "defective replication virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and is unable to produce offspring. In certain embodiments, the sole AAV sequence is the AAV reverse terminal repeat (ITR), which is typically located at the 5' and 3' ends of the vector genome to allow genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0026] As used herein, “vector genome” refers to a nucleic acid sequence packaged inside the rAAV capsid that forms a viral particle. Such a nucleic acid sequence includes an AAV reverse terminal repeat (ITR). In the examples herein, the vector genome includes, at least, an AAV5'ITR, a coding sequence(s), and an AAV3'ITR from 5' to 3'. ITRs other than those from AAV2, AAV from a different source than the capsid, or full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides rep function or trans-complementary AAV during production. Further other ITRs may be used. In addition, the vector genome includes regulatory sequences that direct the expression of gene products. Preferred components of the vector genome are discussed in more detail herein.

[0027] rAAV consists of an AAV capsid and a vector genome. The AAV capsid is a collection of heterogeneous populations of vp1, vp2, and vp3 proteins. As used herein, when used to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof refers to a population of non-identical elements having, for example, vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0028] As used herein, the term “heterogeneous population” as used in relation to the vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to the differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1, vp2, and vp3 proteins, which have modifications from the expected amino acid residues. These subpopulations contain at least certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in the asparagine-glycine pair, and optionally further contain other deamidated amino acids, where deamidation results in amino acid changes and any other modifications.

[0029] As used herein, a “subpopulation” of vp proteins means, unless otherwise specified, a group of vp proteins that share at least one defined common feature and consist of at least one group member and fewer members than all members of the reference group. For example, a “subpopulation” of vp1 proteins is, unless otherwise specified, at least one (1)vp1 protein in an assembled AAV capsid, and may be less than all vp1 proteins. A “subpopulation” of vp3 proteins is, unless otherwise specified, one (1)vp3 protein, and may be fewer than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins may be a subpopulation of vp proteins, vp2 proteins may be a distinct subpopulation of vp proteins, and vp3 may be a further subpopulation of vp proteins in an assembled AAV capsid. In another example, the vp1, vp2, and vp3 proteins may comprise subpopulations having different modifications, for example, at least one, two, three, or four highly deamidated asparagines, such as asparagine-glycine pairs.

[0030] Unless otherwise specified, high deamidation is defined as at least 45% deamidation at the reference amino acid position, compared to the predicted amino acid sequence at the reference amino acid position, and at least 50% deamidation at the reference amino acid position. This refers to % 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 approximately 100% deamidation (for example, at least 80% of asparagine at amino acid 57 of SEQ ID NO: 2 may be deamidated based on total vp1 protein, and may be deamidated based on total vp1, vp2, and vp3 proteins). Such percentages may be determined using 2D gel, mass spectrometry, or other preferred techniques.

[0031] While not intended to be theoretically bound, deamidation of at least highly deamidated residues in vp proteins within AAV capsids is considered to be primarily non-enzymatic, driven by functional groups within the capsid protein that deamidate selected asparagine, and to a lesser extent, glutamine residues. Efficient capsid assembly of most 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 unaffected by assembly dynamics. Generally, extensive deamidation in the VP1-specific (VP1-u) region (~aa1-137), which is thought to be located internally before cell entry, suggests that VP deamidation may occur before capsid assembly.

[0032] While not intended to be theoretically binding, the deamidation of N may occur by nucleophilic attack on the carbon atom of the side-chain amide group of Asn via the skeletal nitrogen atom of its C-terminal residue. An intermediate ring-closed succinimide residue is thought to be formed. This succinimide residue then undergoes rapid hydrolysis to yield the final product, aspartic acid (Asp) or isoaspartic acid (IsoAsp). Therefore, in certain embodiments, the deamidation of asparagine (N or Asn) yields Asp or IsoAsp, which can be interconverted via succinimide intermediates, for example, as illustrated below. [ka]

[0033] As provided herein, each deamidated N in VP1, VP2, or VP3 may independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and / or interconversion blends of Asp and isoAsp, or combinations thereof. Any suitable ratio of α- and isoaspartic acid may exist. For example, in certain embodiments, the ratio may be asparagine to isoasparagine in a ratio of 10:1 to 1:10, asparagine to isoasparagine in a ratio of about 50:50, or asparagine to isoasparagine in a ratio of about 1:3, or another selected ratio.

[0034] In certain embodiments, one or more glutamines (Q) are glutamic acid (Glu), These can be deamidated to α-glutamic acid, γ-glutamic acid (Glu), or a blend of α- and γ-glutamic acid, and interconverted via a common glutalimid intermediate. Any preferred ratio of α- and γ-glutamic acid may exist. For example, in certain embodiments, the ratio may be α:γ in an α:1 to 1:10 ratio, α:γ in an α:γ ratio of about 50:50, or α:γ in an α:γ ratio of about 1:3, or another selected ratio. [ka]

[0035] Therefore, rAAV contains subpopulations in the rAAV capsid of vp1, vp2, and / or vp3 proteins having deamidated amino acids, comprising at least one subpopulation containing at least one highly deamidated asparagine. Furthermore, other modifications may include isomerization at particularly selected aspartic acid (D or Asp) residue positions. In yet another embodiment, modifications may include amidation at the Asp position.

[0036] In certain embodiments, the AAV capsid comprises subpopulations of vp1, vp2, and vp3 having at least 1, at least 2, at least 3, at least 4, at least 5 to at least about 25 deamidated amino acid residue positions, of which at least 1–10%, at least 10–25%, at least 25–50%, at least 50–70%, at least 70–100%, at least 75–100%, at least 80–100%, or at least 90–100% are deamidated compared to the amino acid sequence encoding the vp protein. The majority of these may be N residues. However, Q residues may also be deamidated.

[0037] As used herein, “encoded amino acid sequence” refers to the amino acids predicted based on the translation of known DNA codons in a reference nucleic acid sequence that are translated into amino acids. The following table illustrates DNA codons and 20 common amino acids, showing both single-letter codes (SLC) and three-letter codes (3LC). [Table 1]

[0038] In certain embodiments, the rAAV has an AAV capsid having vp1, vp2, and vp3 proteins having subpopulations containing combinations of 2, 3, 4, 5 or more deamidation residues at the positions shown in the table provided herein and incorporated herein by reference.

[0039] Deamidation of rAAV can be determined using 2D gel electrophoresis and / or mass spectrometry and / or protein modeling techniques. Online chromatography can be performed on 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 are acquired using the data-dependent Top-20 method of the Q Exactive HF, dynamically selecting the most abundant yet unsequenced precursor ions from a survey scan (200–2000 m / z). Sequencing is performed via higher-energy collisional dissociation fragmentation at a target value of 1e5 ions determined by predictive auto-increase control, with precursor isolation performed in a 4 m / z window. Survey scans were acquired at a resolution of 120,000 at m / z 200. HCD spectral resolution can be set to 30,000 at m / z 200 with a maximum ion implantation time of 50 ms and a normalized collision energy of 30. The S-lens RF level can be set to 50 to optimize the transmittance of the m / z region occupied by peptides from the digest. Precursor ions can be excluded in six or more charge states from single, unassigned, or fragmentation selection. BioPharma Finder 1.0 software (Thermo Fischer Scientific) can be used to analyze the acquired data. For peptide mapping, a single-entry protein FASTA database with carbamide methylation set as the fixed modification, oxidation, deamide, and phosphorylation set as variable modifications, 10 ppm mass precision, high protease specificity, and MS / MS spectroscopy are available. The search is performed using a confidence level of 0.8. Suitable proteases may include, for example, trypsin or chymotrypsin. Since deamidation adds the mass of the intact molecule + 0.984 Da (mass difference of -OH and -NH2 groups), the mass spectrometric identification of deamidated peptides is relatively straightforward. The percentage of deamidation of 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. Given the number of possible deamidation sites, isotonic species deamidated at different sites may co-migrate in a single peak. Therefore, multiple deamidation sites can be identified or distinguished using fragment ions derived from peptides with multiple potential 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 is the same for all isomeric species and that they are independent at the deamidation sites. It will be understood by those skilled in the art that several variations of these exemplary methods may be used. For example, suitable mass spectrometers may include quadrupole time-of-flight mass spectrometers (QTOF) such as the Waters Xevo or Agilent 6530, or orbitrap devices such as Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Suitable liquid chromatography systems include, for example, the Acquity UPLC system from Waters or Agilent systems (1100 or 1200 series). Suitable data analysis software includes, for example, MassLynx (Waters), Pinpoint and Pepfinder (Thermo Fischer Scientific), Mascot (Matrix Science), and Peaks DB (Bioinformatics Solutions). Further techniques include, for example, X.Jin, published online on June 16, 2017. This can be described in et al, Hu Gene Therapy Methods, Vol.28, No.5, pp.255-267.

[0040] In addition to deamidation, other modifications may occur, but one amino acid will not be converted to a different amino acid residue. Such modifications may include acetylation, isomerization, phosphorylation, or oxidation.

[0041] Modification of deamidation: In certain embodiments, the AAV is modified to reduce deamidation by changing the glycine in the asparagine-glycine pair. In other embodiments, asparagine is changed to a different amino acid, such as glutamine, which deamidates more slowly, or 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, amino acids lacking an amide or amine side group refer to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, phenylalanine, tyrosine, or tryptophan, and / or proline. The modifications described may be on one, two, or three of the asparagine-glycine pairs found in the encoded AAV amino acid sequence. In certain embodiments, such modifications are not made on all four asparagine-glycine pairs. Therefore, methods for reducing deamidation of AAV and / or manipulated AAV variants have a lower deamidation rate. In addition, or alternatively, deamidation of AAV can be reduced by changing one or more other amide amino acids to non-amide amino acids. In certain embodiments, the mutant AAV capsids described herein include mutations in the asparagine-glycine pair such that glycine is changed to alanine or serine. The mutant AAV capsid may include one, two, or three mutations in which the reference AAV naturally contains four NG pairs. In certain embodiments, the AAV capsid may include one, two, three, or four such mutations in which the reference AAV naturally contains five NG pairs. In certain embodiments, the mutant AAV capsid includes only a single mutation in the NG pair. In certain embodiments, mutation A The AV capsid contains mutations in two distinct NG pairs. In certain embodiments, the mutant AAV capsid is two distinct NG pairs containing mutations located at structurally distinct positions within the AAV capsid. In certain embodiments, the mutations are not in the VP1 intrinsic region. In certain embodiments, one of the mutations is in the VP1 intrinsic region. Optionally, the mutant AAV capsid does not contain modifications within the NG pairs but contains mutations to minimize or eliminate deamidation in one or more asparagine or glutamine located outside the NG pairs.

[0042] In certain embodiments, a method is provided to increase the potency of an rAAV vector, which involves manipulating an AAV capsid to eliminate one or more NGs in the wild-type AAV capsid. In certain embodiments, the coding sequence of the "G" in "NG" is manipulated to code for a different amino acid. In the following specific examples, "S" or "A" is substituted. However, other preferred amino acid coding sequences may be selected. For example, based on the AAV8 numbering, see the following table for coding sequences for at least one of the following positions: N57+1, N263+1, N385+1, N514+1, N540+1 are modified or shown in the table below. In certain embodiments, the AAV8 mutation avoids altering the NG pair at positions N57, N94, N263, N305, Q467, N479, and / or N653. In certain embodiments, other AAVs use AAV8 numbering as a reference to avoid mutations at the corresponding N positions, which are determined based on alignment with AAV8.

[0043] These amino acid modifications can be performed by conventional genetic engineering techniques. For example, a nucleic acid sequence containing modified AAV vp codons may be generated, in which one to three of the glycine-coding codons in the arginine-glycine pair are modified to encode an amino acid other than glycine. In certain embodiments, a nucleic acid sequence containing modified arginine codons may have one to three of the arginine-glycine pairs manipulated, resulting in the modified codon encoding an amino acid other than arginine. Each modified codon may encode a different amino acid. Alternatively, one or more of the modified codons may encode the same amino acid. In certain embodiments, these modified AAVrh79, AAV8.AR2.08, or AAV5.5.9 nucleic acid sequences may be used to generate mutant rAAVs having capsids with lower deamidation than the natural AAVrh79, AAV8.AR2.08, or AAV5.5.9 capsids. Such mutated rAAVs may have reduced immunogenicity and / or increased stability during storage, particularly in suspension form.

[0044] This specification also provides nucleic acid sequences encoding AAV capsids having reduced deamidation. Designing nucleic acid sequences encoding these AAV capsids, including DNA (genomic or cDNA) or RNA (e.g., mRNA), is within the scope 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 various methods. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or by companies providing codon optimization services, such as DNA2.0 (Menlo Park, CA). One codon optimization method is described, for example, in International Patent Publication WO 2015 / 012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication 2014 / 0032186 and U.S. Patent Publication 2006 / 0136184. Preferably, the entire open reading frame (ORF) of the product is modified. However, in some embodiments, only fragments of the ORF may be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence to produce nucleic acid fragments of codon-optimized coding regions that encode the polypeptide. Several options are available for performing actual modifications to codons or for synthesizing codon-optimized coding regions designed as described herein. Such modifications or synthesis are standard and routine molecules well known to those skilled in the art. This can be done using biological manipulation. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides long and spanning the desired sequence length, are synthesized by standard methods. These oligonucleotide pairs are synthesized so that, upon annealing, they form 80-90 base pair double-stranded fragments containing aggregated ends, for example, each oligonucleotide in a pair is synthesized to extend by 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 oligonucleotide pair is designed to anneal with the single-stranded end of another oligonucleotide pair. The oligonucleotide pairs are annealed, and then approximately 5-6 of these double-stranded fragments are annealed together via aggregated single-stranded ends, then ligated together, and cloned into a standard bacterial cloning vector, e.g., the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs, consisting of 5-6 fragments of 80-90 base pair fragments ligated together, i.e., fragments of approximately 500 base pairs, are prepared so that the entire desired sequence is represented in a series of plasmid constructs. The insertions in these plasmids are then cleaved 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.

[0045] In certain embodiments, an AAV capsid is provided having a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) containing multiple highly deamidated "NG" sites. In certain embodiments, the highly deamidated sites are located at the positions shown below, with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified so that the referenced "NG" is removed, and the mutant "NG" is manipulated to a different position.

[0046] In certain embodiments, a mixed population of rAAVs arises from a production system using a single AAV capsid nucleic acid sequence encoding a predicted AAV VP1 amino acid sequence for one AAV type. However, the manufacturing and production processes provide a heterogeneous population of the capsid proteins described above.

[0047] In certain embodiments, a novel isolated AAVrh79 capsid is provided. The nucleic acid sequence encoding AAV is provided in SEQ ID NO: 1, and the encoded amino acid sequence is provided in SEQ ID NO: 2.

[0048] In certain embodiments, rAAV comprises an AAVrh79 capsid. The AAVrh79 capsid comprises a heterogeneous population of AAVrh79 vp1 protein, AAVrh79 vp2 protein, and AAVrh79 vp3 protein. In one embodiment, the AAVrh79 capsid is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 1–738. Optionally, the vp3 protein from a nucleic acid sequence excluding the vp1-specific region (approximately aa1–137) or the vp2-specific region (approximately aa1–203), the vp1 protein produced from SEQ ID NO: 1, or a sequence co-expressing the vp1 protein is produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 1, encoding the predicted amino acid sequence of SEQ ID NO: 1–738. In other embodiments, the AAVrh79 vp2 protein is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately amino acids 138-738 of SEQ ID NO: 2, the vp2 protein is produced from a sequence containing at least nucleotides 412-2214 of SEQ ID NO: 1, or from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately amino acids 138-738 of SEQ ID NO: 2, which is at least 70% identical to at least nucleotides 412-2214 of SEQ ID NO: 1. The vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 204-738 amino acids of SEQ ID NO: 2, the AAVrh79 vp3 protein produced from a sequence containing at least nucleotides 610-2214 of SEQ ID NO: 1, or the vp3 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 204-738 amino acids of SEQ ID NO: 2, which is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO: 1.

[0049] In certain embodiments, the AAVrh79 capsid comprises a heterogeneous population of vp1 proteins, which are products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; a heterogeneous population of vp2 proteins, which are products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 at least approximately amino acids 138-738; and a heterogeneous population of vp3 proteins, which are products of a nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO: 2.

[0050] The AAVrh79 vp1, vp2, and vp3 proteins comprise a subpopulation with amino acid modifications containing at least two highly deamidated asparagine (N) in the asparagine-glycine pair of SEQ ID NO: 2, and optionally further comprising subpopulations containing other deamidated amino acids, where deamidation results in amino acid changes. Compared to the number in SEQ ID NO: 2, high levels of deamidation are observed in NG pairs N57, N263, N385, and / or N51. Deamidation has been observed in other residues as shown in the table and examples below. In certain embodiments, AAVrh79 may be deamidated and / or methylated (e.g., ~R487) (typically less than 5% of a given residue, more typically less than 1%), isomerized (e.g., at D97) (typically less than 5% of a given residue, more typically less than 1%), or phosphorylated (e.g., about 10-60% if present, or about 10-30%, or about 20%). It may have other modifications, including oxidation (e.g., in the range of approximately 60%) (for example, one or more of S149, ~S153, ~S474, ~T570, ~S665), or oxidation (for example, one or more of W248, W307, W307, M405, M437, M473, W480, W480, W505, M526, M544, M561, W621, M637, and / or W697). Optionally, W may be oxidized to kynurenine. [Table 2]

[0051] In certain embodiments, the AAVrh79 capsid is modified at one or more of the positions identified in the table above, determined by mass spectrometry using trypsinase, within the range shown below. In certain embodiments, one or more of the following positions, or glycine following N, are modified as described herein. Residue numbers are based on the AAVrh79 sequence provided herein. See Sequence ID No. 2.

[0052] In certain embodiments, the nucleic acid sequence encoding the AAVrh79 vp1 capsid protein is provided in SEQ ID NO: 1. In other embodiments, a nucleic acid sequence 70% to 99.9% identical to SEQ ID NO: 1 may be selected to express the AAVrh79 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: 1. However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 2 may be selected for use in the production of the rAAV capsid. In certain embodiments, the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 1, or a sequence that is at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 1, and encodes SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence has at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nucleic acid sequence of SEQ ID NO: 1, or at least nt412 to about nt2214 of SEQ ID NO: 1, and encodes the vp2 capsid protein (about aa138 to 738) of SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence has at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nucleic acid sequence of about nt610 to about nt2214 of SEQ ID NO: 1, or at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nt of SEQ ID NO: 1, and encodes the vp3 capsid protein (about aa204 to 738) of SEQ ID NO: 2.

[0053] The present invention also comprises a nucleic acid sequence encoding mutant AAVrh79, wherein one or more residues are modified to reduce deamidation or other modifications as specified herein. Such nucleic acid sequences can be used in the production of mutant rAAVrh79 capsids.

[0054] In certain embodiments, a novel AAV8.AR2.08 capsid is provided. The nucleic acid sequence encoding AAV is provided in SEQ ID NO: 17, and the encoded amino acid sequence is provided in SEQ ID NO: 18. In one embodiment, recombinant adeno-associated virus (rAAV) has the AAV8.AR2.08 capsid. Alignments of the amino acid sequences of AAV8T, AAV8.AR2.08, and AAV8 are provided in Figure 13A. Alignments of the nucleic acid sequences of AAV8T, AAV8.AR2.08, and AAV8 are provided in Figures 13B-13D.

[0055] In certain embodiments, the AAV8.AR2.08 capsid is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 18 from nucleotides 1 to 738, a vp1 protein produced from SEQ ID NO: 17, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 17 encoding the predicted amino acid sequence of SEQ ID NO: 18 from nucleotides 1 to 738, an AAV8.AR2.08 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 18 from nucleotides 138 to 738, a vp2 protein produced from a sequence containing at least nucleotides 412 to 2214 of SEQ ID NO: 17, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO: 17 encoding the predicted amino acid sequence of SEQ ID NO: 18 from nucleotides 138 to 738, or at least approximately 2 The AAV8.AR2.08 capsid protein includes an AAV8.AR2.08 vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 04-738, a vp3 protein produced from a sequence containing at least nucleotides 610-2214 of SEQ ID NO: 17, or a vp3 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 204-738 of SEQ ID NO: 18, which is at least 70% identical to at least nucleotides 610-2214 of SEQ ID NO: 17.

[0056] Additionally, or alternatively, the AAV8.AR2.08 capsid comprises a heterogeneous population of vp1 protein, which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18; a heterogeneous population of vp2 protein, which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 at least approximately amino acids 138-738; and a heterogeneous population of vp3 protein, which is the product of the nucleic acid sequence encoding at least amino acids 204-738 of SEQ ID NO: 18, wherein the vp1, vp2, and vp3 proteins comprise subpopulations having amino acid modifications including at least two highly deamidated asparagine (N) in the asparagine-glycine pair of SEQ ID NO: 18, and optionally further comprising subpopulations containing other deamidated amino acids, where deamidation results in amino acid changes. AAV8.AR2.08 is characterized by having highly deamidated residues at positions N57, N263, N385, N514, and N540, for example, based on the numbering of AAV8.AR2.08 VP1 [SEQ ID NO: 18]. Additionally, the residues at the positions in the following table and the detailed table in the application indicate the deamidation observed in the AAV8.AR2.08 capsid.

[0057] In certain embodiments, the AAV8.AR2.08 capsid is modified at one or more of the following positions, within the range provided below, as determined by mass spectrometry using trypsinase. In certain embodiments, one or more of the following positions, or glycine following N, are modified as described herein. For example, in certain embodiments, G may be modified to S or A at, for example, position 58, 264, 386, 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, N in the NG pair is modified to Q while retaining G. In certain embodiments, both amino acids in the NG 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.

[0058] In addition to deamidation, other modifications may include isomerization (e.g., with one or more of D442 and / or D584) (1-15%), phosphorylation (e.g., with one or more of ~S149, ~T417, ~T454, ~T493, S600, and / or ~T663), and / or oxidation (e.g., with one or more of ~W22, ~M204, ~M212, W248, W307, M405, M437, M473, W480, W505, M526, M561, M607, ~W609, W621, M637, W697). Further positions may have these or other modifications (e.g., acetylation or further deamidation). [Table 3]

[0059] In certain embodiments, the nucleic acid sequence encoding the AAV8.AR2.08 vp1 capsid protein is provided in SEQ ID NO: 17. In other embodiments, a nucleic acid sequence 70% to 99.9% identical to SEQ ID NO: 17 may be selected to express the AAV8.AR2.08 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: 17. However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 18 may be selected for use in the production of the rAAV8.AR2.08 capsid. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 17, or a sequence that is 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: 17, and encodes SEQ ID NO: 18. In certain embodiments, the nucleic acid sequence has at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nucleic acid sequence of SEQ ID NO: 17, or at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nucleic acid sequence of SEQ ID NO: 17, or at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nt of SEQ ID NO: 17, and encodes the vp3 capsid protein of SEQ ID NO: 18.

[0060] The present invention also comprises a nucleic acid sequence encoding the mutant AAV8.2.08, and one or more residues The nucleic acid sequence has been modified to reduce deamidation or other modifications as specified herein. Such nucleic acid sequences can be used in the production of mutant rAAV8.2.08.

[0061] In certain embodiments, a novel AAV5.5.9 capsid is provided. The nucleic acid sequence encoding AAV is provided in SEQ ID NO: 9, and the encoded amino acid sequence is provided in SEQ ID NO: 10. The amino acid sequence alignments of AAV5.5.9, AAV9, and AAVPHP.B are shown in Figure 12A. The nucleic acid sequence alignments of AAV5.5.9, AAV9, and AAVPHP.B are shown in Figures 12B-12E. In one embodiment, recombinant adeno-associated virus (rAAV) is a heterogeneous population of AAV5.5.9 vp1 protein produced by expression from the nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 10 (1-726), vp1 protein produced from SEQ ID NO: 9, or vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 9 (encoding the predicted amino acid sequence of SEQ ID NO: 1) and AAV5.5.9 produced by expression from the nucleic acid sequence encoding the predicted amino acid sequence of at least approximately amino acids 137-726 of SEQ ID NO: 10. The AAV5.5.9 capsid contains a heterogeneous population of vp2 proteins, including a vp2 protein produced from a sequence containing at least nucleotides 409-2178 of SEQ ID NO: 9, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 577-2178 of SEQ ID NO: 9 and encodes the predicted amino acid sequence of at least approximately amino acids 137-726 of SEQ ID NO: 10; and an AAV5.5.9 capsid protein produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least approximately amino acids 193-726 of SEQ ID NO: 10, including a heterogeneous population of vp3 proteins produced from a sequence containing at least nucleotides 577-2178 of SEQ ID NO: 9, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 577-2178 of SEQ ID NO: 9 and encodes the predicted amino acid sequence of at least approximately amino acids 193-726 of SEQ ID NO: 10.

[0062] Additionally, or alternatively, the AAV5.5.9 capsid comprises a heterogeneous population of vp1 protein, which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10; a heterogeneous population of vp2 protein, which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10 at least approximately amino acids 137–726; and a heterogeneous population of vp3 protein, which is the product of the nucleic acid sequence encoding at least amino acids 193–726 of SEQ ID NO: 10, wherein the vp1, vp2, and vp3 proteins comprise subpopulations having amino acid modifications including at least two highly deamidated asparagine (N) in the asparagine-glycine pair of SEQ ID NO: 10, and optionally further comprising subpopulations containing other deamidated amino acids, where deamidation results in amino acid changes. [Table 4]

[0063] In certain embodiments, the nucleic acid sequence encoding the AAV5.5.9 vp1 capsid protein is provided in SEQ ID NO: 9. In other embodiments, a nucleic acid sequence that is 70% to 99.9% identical to SEQ ID NO: 9 may be selected to express the AAV5.5.9 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: 9. However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 10 may be selected for use in the production of the rAAV5.5.9 capsid. In certain embodiments, the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 10, or a sequence that is at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 9, and encodes SEQ ID NO: 10. In certain embodiments, the nucleic acid sequence has at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical sequences to the nucleic acid sequence of SEQ ID NO: 9, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99%, of the VP2 capsid protein (at least AA137 to AA726) of SEQ ID NO: 10. In certain embodiments, the nucleic acid sequence is the nucleic acid sequence of approximately nt577 to approximately nt2178 of SEQ ID NO: 9, or has a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical to nt577 to nt2178 of SEQ ID NO: 9, and encodes the vp3 capsid protein (approximately aa193 to 726) of SEQ ID NO: 10.

[0064] The present invention also comprises a nucleic acid sequence encoding mutant AAV5.5.9, wherein one or more residues are modified to reduce deamidation or other modifications as identified herein. Such nucleic acid sequences can be used in the production of mutant rAAV5.5.9.

[0065] I.rAAV vector As described above, the novel AAV sequences and proteins are useful for rAAV production and for recombinant AAV vectors that may 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.

[0066] The genomic sequence packaged in the AAV capsid and delivered to the host cell typically consists of at least the transgene, its regulatory sequence, and the AAV reverse terminal repeat (ITR). Both single-stranded AAV and self-complementary (sc)AAV are included in rAAV. The transgene is a nucleic acid coding sequence heterogeneous to the vector sequence that encodes the polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably ligated to the regulatory element in a manner that enables transcription, translation, and / or expression of the transgene in the cells of the target tissue.

[0067] The AAV sequence of the vector typically contains cis-acting 5′ and 3′ reverse-terminal repeat sequences (see, e.g., B.J. Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequence is approximately 145 bp long. Preferably, the entire sequence substantially encoding the ITR is used intramolecularly, although some minor modifications to these sequences are permissible. The ability to modify these ITR sequences is within the scope 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)). An example of such molecules used in the present invention is a “cis-acting” plasmid containing a transgene flanked by 5' and 3' AAV ITR sequences, with the selected transgene sequence and associated regulatory elements being adjacent to the 5' and 3' AAV ITR sequences. In one embodiment, the ITR is derived from a different AAV than the one supplying the capsid, resulting in a pseudo-type vector. In one embodiment, the ITR sequence is derived from AAV2. A shortened version of the 5' ITR, referred to as ΔITR, is described, lacking the D sequence and terminal resolution sites (trs). In other embodiments, full-length AAV 5' and 3' ITRs are used. However, ITRs from other AAV origins may be selected. If the ITR source is AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudo-type. However, other configurations of these elements may be preferred.

[0068] In addition to the key elements described above for recombinant AAV vectors, the vector also includes necessary conventional regulatory elements that are operably linked to the transgene in such a manner that they enable its transcription, translation, and / or expression in cells transfected with a plasmid vector or infected with a virus produced by the present invention. The "linked" sequence includes both the gene of interest and adjacent regulatory sequences, as well as regulatory sequences that act in or away from the gene of interest to control it.

[0069] The regulatory element typically includes, for example, a promoter sequence located between a selected 5'ITR sequence and a coding sequence as part of the expression control sequence. Constitutive promoters, regulated promoters [see, e.g., WO2011 / 126808 and WO2013 / 04943], tissue-specific promoters, or promoters responsive to physiological hints may be used in the vectors described herein. The promoter(s) may be selected from different sources, such as the human cytomegalovirus (CMV) pre-initial enhancer / promoter, SV40 initial enhancer / promoter, JC poliomavirus promoter, myelin basic protein (MBP) or collagen fiber acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latent-associated promoter (LAP), Roussarcoma virus (RSV) long-terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and chicken beta-actin promoter. In one embodiment, the promoter is a liver-specific promoter, such as the one referred to herein as LSP.

[0070] In addition to the promoter, the vector may include one or more other suitable transcription start, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly-A) signals, sequences that stabilize cytoplasmic mRNA, e.g., WPRE, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance the 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 includes two or more expression enhancers. These enhancers may be the same or different from one another. For example, the enhancer may include a CMV pre-initial enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the duplicate copies of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further comprises introns, such as chicken beta-actin introns. Other suitable introns include those known in the art, for example, described in International Publication No. 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. An example of such a sequence is a modified WPRE sequence, which can be manipulated upstream of the polyA sequence and downstream of the coding sequence [e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619].

[0071] These rAAVs are particularly suitable for gene delivery for therapeutic and immunization purposes, 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., a protein) can be obtained from the desired culture after transfecting host cells with an rAAV containing a molecule 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.

[0072] Therapeutic transgenes Useful products encoded by the transgene include deletions or replacements of the deletion gene. The range includes various gene products that inactivate, "knock out," or "knock down" genes, reduce the expression of genes expressed at undesirable high levels, or deliver gene products with desired therapeutic effects. In most embodiments, the treatment is "somatic cell gene therapy," i.e., the introduction of genes into somatic cells that do not produce sperm or eggs. In certain embodiments, the transgene-expressing protein has a sequence of a natural human sequence. However, in other embodiments, a synthetic protein is expressed. Such proteins may be intended for the treatment of humans, or in other embodiments, 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 human populations.

[0073] Suitable gene products may include those associated with familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such rare diseases include, among others, spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (e.g., methyl-CpG binding protein 2 (MeCP2), UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia (e.g., frataxin), and progranulin (PRGN) (associated with 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.

[0074] 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), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFα, and activin. This may include, but is not limited to, any one of the transforming growth factor α superfamily including inhibin, or any of the bone morphogenetic proteins (BMPs) BMP1-15, the growth factor heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT-4 / 5, ciliary body neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), any one of neuruturin, agrin, semaphorin / colapsin, netrin 1 and netrin 2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and any one of the tyrosine hydroxylase families.

[0075] Other useful transgene products include thrombopoietin (TPO), interleukins (IL), and IL-1 to IL-36 (e.g., human interleukins). This includes proteins that regulate the immune system, including but not limited to cytokines such as 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 proteins, leukemia inhibitors, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β, and γ, stem cell factors, and lymphokines such as flk-2 / flt3 ligand. Gene products produced by the immune system are also useful in this invention. These include immunoglobulin IgG, This includes, but is not limited to, 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 may 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 proteins (MCPs), complement-mediated analytic proteins (DAFs), CR1, CF2, CD59, and C1 esterase inhibitors (C1-INH).

[0076] Further useful gene products include any one of the following: 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. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD, and myogenin, ETS boxes containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box-binding proteins, interferon regulator (IRF-1), Wilms tumor protein, ETS-binding proteins, STAT, GATA box-binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

[0077] Other useful gene products include carbamoyl synthetase I, ornithine transcarbamylase (OTC), arginosuccinate synthetase, arginosuccinate lyase (ASL) for the treatment of arginosuccinate lyase deficiency, arginase, fumaacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, rhesus monkey alpha-fetoprotein (AFP), rhesus monkey chorionic gonadotropin (CG), glucose-6-phosphatase, porphobilinogen deaminase, and cystathione beta-sin. These include tase, 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 regulator (CFTR) sequences, and dystrophin gene products [e.g., mini- or micro-dystrophin]. Further useful gene products include enzymes that may be useful in enzyme replacement therapy, which are beneficial in various conditions resulting from insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate may be used in the treatment of lysosomal storage disorders (e.g., suitable genes include those encoding β-glucuronidase (GUSB)).

[0078] In certain embodiments, rAAV may be used in a gene editing system, which may involve co-administration of one or more rAAV strains. For example, rAAV may be manipulated to deliver SpCas9, SaCas9, ARCUS, Cpf1, and other suitable gene editing constructs.

[0079] Other useful gene products include hemophilia B (including factor IX) and hemophilia A (factor VIII and its variants, such as heterodimers and B-deletion domains). This includes light and heavy chains; including those used in the treatment of hemophilia (U.S. Patents 6,200,560 and 6,221,349). In some embodiments, the minigene includes the first 57 base pairs of the factor VII heavy chain encoding a 10-amino acid signal sequence, as well as a human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the minigene further includes A1 and A2 domains, as well as 5 amino acids from the N-terminus of the B domain and / or 85 amino acids from the C-terminus of the B domain, as well as A3, C1, and C2 domains. In yet another embodiment, the nucleic acids encoding the factor VIII heavy and light chains are provided within a single minigene separated by 42 nucleic acids encoding 14 amino acids of the B domain (U.S. Patent 6,200,560).

[0080] Other useful gene products include non-natural polypeptides such as chimeric or hybrid polypeptides that have non-natural amino acid sequences, including insertions, deletions, or amino acid substitutions. For example, single-stranded immunoglobulins may be useful in certain immunocompromised patients. Other types of non-natural gene sequences include catalytic nucleic acids such as antisense molecules and ribozymes, which may be used to reduce the overexpression of targets.

[0081] Reducing and / or regulating gene expression is particularly desirable for the treatment of hyperproliferative conditions characterized by overgrowth, as well as cancer and psoriasis. Target polypeptides include polypeptides produced exclusively or at higher levels in hyperproliferating cells compared to normal cells. Target antigens include oncogenes such as myb, myc, and fyn, as well as polypeptides encoded by translocation genes bcr / abl, ras, src, P53, neu, trk, and EGRF. In addition to oncogene products as target antigens, target polypeptides for anticancer 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 the monoclonal antibody 17-1A and folate-binding polypeptides.

[0082] 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 autoimmune-related targets, including cell receptors and cells that produce "self"-targeted 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 an endogenous antigen and initiates an inflammatory cascade associated with the autoimmune disease.

[0083] Further exemplary genes that can be delivered via rAAV include, but are not limited to, glucose-6-phosphatase associated with glycogen storage disorder 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-keto acid dehydrogenase associated with maple syrup urine disease, fumarylacetoacetate hydrolase associated with tyrosinemia type 1, methylmalonyl-CoA mutase associated with methylmalonic acidemia, medium-chain acyl-CoA dehydrogenase associated with medium-chain acetyl-CoA deficiency, and orni Ornithine transcarbamylase (OTC) associated with tin transcarbamylase deficiency, argininosuccinate synthetase (ASS1) associated with citrullinemia, lecithin-cholesterol acyltransferase (LCAT) deficiency, amethylmalonic 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-Nadger disease, adenosine deaminase associated with severe combined immunodeficiency, hypoxanthine guanine phosphoribosyltransferase associated with gout and Leschnyan syndrome, biothymidase associated with biothymidase deficiency, and 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, peroxisome membrane protein 70kDa associated with Zellweger syndrome, arylsulfatase 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, and survival motor neurons associated with spinal muscular atrophy. (SMN) protein, ceramidase associated with Faber lipogranulomatosis, β-hexosaminidase associated with GM2 gangliosidosis and Taysachs and Sandhoff disease, aspartyl-glucosaminidase associated with aspartyl-glucosaminuria, α-fucosidase associated with fucosidosis, α-mannosidase associated with alpha-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 for the treatment of ischemic disease, angiopoietin-1,These include fibroblast growth factors, such as thrombomodulin and tissue factor pathway inhibitors for the treatment of occluded blood vessels seen in atherosclerosis, thrombosis, or embolism; aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; beta-adrenergic receptors, antisense or variants of phospholamban, myocardial (endoplasmic reticulum) adenosine triphosphatase-2 (SERCA2) and cardiac adenylyl cyclase for the treatment of congestive heart failure; tumor suppressor genes such as p53 for the treatment of various cancers; cytokines such as one of various interleukins for the treatment of inflammation and immune disorders and cancer; dystrophin or mini-dystrophin and eutrophin or mini-trophin for the treatment of muscular dystrophy; and insulin or GLP-1 for the treatment of diabetes.

[0084] Further target genes and diseases include, for example, dystonin gene-related disorders such as hereditary sensory and autonomic dysfunction type VI (the DST gene encodes dystonin, and depending on the size of the protein (~7570aa), a dual AAV vector may be required, deletion of a functional variant that prevents the sensation of pain, and acquisition of a functional variant that causes pain, such as erythromelagia, and SCN9A-related diseases). Another condition is Charcot-Marie-Tooth types 1F and 2E, caused by mutations in the NEFL gene (neuronal filament light chain), characterized by progressive peripheral motor and sensory neuropathy with variable clinical and electrophysiological expression.

[0085] In certain embodiments, the rAAVs described herein may be used in the treatment of mucopolysaccharidosis (MPS) disorders. Such rAAVs include nucleic acid sequences encoding α-L-iduronidase (IDUA) for the treatment of MPS I (Hurler, Hurler-Schaye, and Schaye syndromes), nucleic acid sequences encoding iduronate-2-sulfatase (IDS) for the treatment of MPS II (Hunter syndrome), and MPS III A, B, C, and D Nucleic acid sequences encoding sulfamidase (SGSH) for treating Sanfilippo syndrome, MPS IV A and B (Morcio syndrome), MPS This may include carrying a nucleic acid sequence encoding arylsulfatase B (ARSB) for the treatment of MPS VI (Maroto-Lamy syndrome), a nucleic acid sequence encoding hyaluronidase for the treatment of MPS IX (hyaluronidase deficiency), and a nucleic acid sequence encoding beta-glucuronidase for the treatment of MPS VII (Sly syndrome).

[0086] Immunogenic transgenes In some embodiments, rAAV vectors containing nucleic acids encoding cancer-related gene products (e.g., tumor suppressors) may be used to treat cancer by administering rAAV containing the rAAV vector to subjects with cancer. In some embodiments, rAAV vectors containing nucleic acids encoding small interfering nucleic acids (e.g., shRNA, miRNA) that inhibit the expression of cancer-related gene products (e.g., oncogenes) may be used to treat cancer by administering rAAV containing the rAAV vector to subjects with cancer. In some embodiments, rAAV vectors containing nucleic acids encoding cancer-related gene products (or functional RNAs that inhibit the expression of cancer-related genes) may be used for research purposes, such as studying cancer or identifying therapeutic agents to treat cancer. The following is a non-restrictive 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, CBFB, CBLB, CCL2, CCN D1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC 2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG , CENPC1, CGRRF1, CHAF1A, CIB1, 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、F GFR1、FGR、FKBP8、FN1、FOS、FOSL1、FOSL2、FOXG1A、FOXO1A、FRAP1、FRZB、FTL、FZD2、FZD5、F ZD9、G22P1、GAS6、GCN5L2、GDF15、GNA13、GNAS、GNB2、GNB2L1、GPR39、GRB2、GSK3A、GSPT1、GT F2I、HDAC1、HDGF、HMMR、HPRT1、HRB、HSPA4、HSPA5、HSPA8、HSPB1、HSPH1、HYAL1、HYOU1、ICA M1、ID1、ID2、IDUA、IER3、IFITM1、IGF1R、IGF2R、IGFBP3、IGFBP4、IGFBP5、IL1B、ILK、ING1、 IRF3、ITGA3、ITGA6、ITGB4、JAK1、JARID1A、JUN、JUNB、JUND、K-ALPHA-1、KIT、KITLG、KLK10 、KPNA2、KRAS2、KRT18、KRT2A、KRT9、LAMB1、LAMP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF、LYN、 LZTR1, MADH1, MAP2K2, MAP3K8, MAPK12, MAPK13, MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3, MYB, MYBL1, MYBL2, MYC MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、NF1、NF2、NFKB1、NFKB2、NFSF7、NID、NINE、NMBR、NME1、NME2、NME3、NO TCH1、NOTCH2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、NSEP1、OSM、PA2G4、PABPC1、PCNA、PCTK 1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PEA15、PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、 PIM1、PKM2、PKMYT1、PLK2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP、PR SS15、PSMA1、PTCH、PTEN、PTGS1、PTMA、PTN、PTPRN、RAB5A、RAC1、RAD50、RAF1、RALBP1、RAP1A、RARA、RARB、RA SGRF1、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、SIVATP53 ) , SNAI2 , SND1 , SNRPB2 , SOCS1 , SOCS3 , SOD1 , SORT1 , SPINT2 , SPRY2 , SRC , SRPX , ST AT1, STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP 1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, T K1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TN K1, TOB1, TP53, TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101 TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1 NT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9.

[0087] rAAV vectors may contain nucleic acids encoding proteins or functional RNAs that regulate apoptosis as transgenes. The following is a non-limiting list of apoptosis-related genes, products of these genes and their homologs, and nucleic acids encoding small interfering nucleic acids (e.g., shRNA, miRNA) that are useful as transgenes in specific embodiments of the present invention, inhibiting the expression of these genes and their homologs: RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, B FAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD4 5A. F21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5.

[0088] Useful transgene products 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 RNA (mRNA). miRNAs are typically expressed naturally as the final 19–25 untranslated RNA product. miRNAs exhibit their activity through sequence-specific interactions with the 3′ untranslated region (UTR) of target mRNA. These endogenously expressed miRNAs form hairpin precursors, which are then processed into miRNA double-stranded and further into “mature” single-stranded miRNA molecules. This mature miRNA induces the miRISC multiprotein complex, which, based on complementarity with the mature miRNA, identifies, for example, a target site on the target mRNA within the 3′ UTR region.

[0089] The following non-restrictive list of miRNA genes and their homologs is useful as a target for small interfering nucleic acids encoded by transgenes or transgenes in specific embodiments of the following methods (e.g., miRNA sponges, antisense oligonucleotides, TuD RNA): 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-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*, hsa-let-71, hsa-let-1 et-71*, hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hsa-miR-1 06a, 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-miR-1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-1207-3p, hsa-miR-12 07-5p, 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, hsa-m iR-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-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p, hsa-mi R-125a-5p、hsa-miR-125b、hsa-miR-125b-1*、hsa-miR-125b-2*、hsa-miR -126、hsa-miR-126*、hsa-miR-1260、hsa-miR-1261、hsa-miR-1262、hsa-m iR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267, hsa-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-miR-1275 hsa-miR-127-5p hsa-miR-1276 hsa-miR-1277 hsa-miR-1278 h sa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282, hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1 287, 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, hs a-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, hsa-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-136*, 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*、hsa-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-154, 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-18 1a*、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-185*、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-1 92, 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 h sa-miR-200c*, hsa-miR-202, hsa-miR-202*, hsa-miR-203, hsa-miR-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*, hs a-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*, hsa-miR-219-1-3p, hsa-miR-219-2-3phsa-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-22 2*, hsa-miR-223, hsa-miR-223*, hsa-miR-224, hsa-miR-23a, hsa-miR-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-m iR-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-3 02b*、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-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30 d、hsa-miR-30d*、hsa-miR-30e、hsa-miR-30e*、hsa-miR-31、hsa-miR-31* 、hsa-miR-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-324-5p 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, hs a-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-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*, hsa-miR-374a, hsa-mi R-374a*、hsa-miR-374b、hsa-miR-374b*、hsa-miR-375、hsa-miR-376a、hs a-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-380 hsa-miR-380* hsa-miR-381 hsa-miR-382 hsa-miR-383 hsa-miR-384hsa-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-m iR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, hsa-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, hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-4 52*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p, hsa-miR-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-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489, hsa-miR-490-3 p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p, hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495, hsa-miR-496, hsa-m iR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p, hsa-miR-499-5p, hsa-miR-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-508-5phsa-miR-509 -3-5p, hsa-miR-509-3p, hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hs a-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*、hsa-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-mi R-519d、hsa-miR-519e、hsa-miR-519e*、hsa-miR-520a-3p、hsa-miR-520a -5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR-520d-3p、hsa-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-526b*, 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-3phsa-miR-5486-5p、hsa-miR-548c-3p、hsa-miR-548c-5p、hsa-miR-548d-3 p、hsa-miR-548d-5p、hsa-miR-548e、hsa-miR-548f、hsa-miR-548g、hsa-m iR-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-5 67, hsa-miR-568, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR-572, hsa-miR-573, 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, hsa-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-601hsa-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-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-mi R-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-mi R-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-641, hsa-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-miR-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* hs a-miR-665, hsa-miR-668, hsa-miR-671-3p, hsa-miR-671-5p, hsa-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-87 4, hsa-miR-875-3p, hsa-miR-875-5p, hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*, hsa-miR-885-3p, hsa-miR-88 5-5p, 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-923, hsa-miR -924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-m iR-934, hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-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 that target the open reading frame 72 (C9orf72) on chromosome 8, which expresses superoxide dismutase (SOD1) associated with amyotrophic lateral sclerosis (ALS), are of interest. ,

[0090] miRNAs inhibit the function of their target mRNA, thereby inhibiting the expression of the polypeptide encoded by that mRNA. Therefore, blocking (partially or completely) the activity of a miRNA (e.g., silencing the miRNA) can effectively induce or restore the expression of the polypeptide whose expression is inhibited (de-repressing the polypeptide). In one embodiment, de-repression of the polypeptide encoded by the mRNA target of a miRNA is achieved by inhibiting intracellular miRNA activity through one of several methods. For example, blocking the activity of a miRNA can be achieved by hybridization with a small interfering nucleic acid (e.g., antisense oligonucleotides, miRNA sponges, TuD RNA) that is complementary or substantially complementary to the miRNA, thereby blocking the interaction between the miRNA and its target mRNA. As used herein, a small interfering nucleic acid substantially complementary to miRNA is a nucleic acid that hybridizes with miRNA and can block the activity of miRNA. In some embodiments, small interfering nucleic acids that are substantially complementary to miRNA are small interfering nucleic acids that are complementary to miRNA in all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases. A “miRNA inhibitor” is a drug that blocks the function, expression, and / or processing of miRNA. Examples of such molecules include, but are not limited to, microRNA-specific antisense, microRNA sponge, tough decoy RNA (TuD RNA), and microRNA oligonucleotides (double-stranded, hairpin, and short oligonucleotides) that inhibit miRNA interaction with the Drosha complex.

[0091] Other useful transgenes may include those encoding immunoglobulins that confer passive immunity to pathogens. “Immunoglobulin molecules” are proteins containing immunoactive portions of immunoglobulin heavy chains and immunoglobulin light chains that are covalently linked to each other and can specifically combine with antigens. Immunoglobulin molecules can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. The terms “antibody” and “immunoglobulin” may be used interchangeably herein.

[0092] An "immunoglobulin heavy chain" is a polypeptide comprising at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of the variable region of the immunoglobulin heavy chain or at least a portion of the constant region of the immunoglobulin heavy chain. Therefore, immunoglobulin-derived heavy chains have regions of significant amino acid sequence homology with members of the immunoglobulin gene superfamily. For example, the heavy chain in the Fab fragment is an immunoglobulin-derived heavy chain.

[0093] An "immunoglobulin light chain" is a polypeptide comprising 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 the immunoglobulin light chain. Therefore, immunoglobulin-derived light chains have regions of significant amino acid homology with members of the immunoglobulin gene superfamily.

[0094] An "immunoadhesin" is a chimeric antibody-like molecule that combines the functional domain of a binding protein, usually a receptor, ligand, or cell adhesion molecule, with an immunoglobulin constant domain, usually including a hinge region and an Fc region.

[0095] A "fragment antigen-binding" (Fab) fragment is a region on an antibody that binds to an antigen. It consists of one constant domain and one variable domain in both the heavy and light chains.

[0096] Antipathogen constructs are selected based on the causative agent (pathogen) of the disease for which protection is required. These pathogens may be of viral, bacterial, or fungal origin, and are used for human diseases. It may be used to prevent infection in non-human mammals or other animals in order to prevent veterinary diseases.

[0097] rAAV may contain genes encoding antibodies, particularly neutralizing antibodies against viral pathogens. Such antiviral antibodies may include anti-influenza antibodies targeted against one or more of influenza A, influenza B, and influenza C. Influenza A viruses are the most virulent human pathogens. Serotypes of influenza A associated with pandemics include H1N1, which caused the Spanish flu of 1918; swine flu of 2009; H2N2, which caused the Asian flu of 1957; H3N2, which caused the Hong Kong flu of 1968; and H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7, which caused avian flu of 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 (smallpox), and vaccinia virus (cowpox) and varicella-zoster virus (pseudorabies), all derived from the poxvirus family. Viral hemorrhagic fevers are caused by members of the arenavirus family (Lassa fever) (a family also associated with lymphocytic choriomeningitis (LCM)), filoviruses (Ebola virus), and hantaviruses (premara). Members of the picornavirus family (a subfamily of rhinoviruses) are associated with the common cold in humans. The coronavirus family includes several non-human viruses, such as infectious bronchitis virus (poultry), porcine gastroenteritis virus (swine), porcine hemagglutinin encephalomyelitis virus (swine), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), and canine coronavirus (dog). Human respiratory coronaviruses are presumed to be associated with 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, rubravirus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, measles and canine distemper, morbilliviruses, 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 (EX, AD7, ARD, OB). Therefore, in certain embodiments, the rAAV vectors described herein may 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, against bacterial pathogens. Neutralizing antibody constructs may also be selected for use in the present invention. In one embodiment, the neutralizing antibody construct is directed against the bacteria themselves. In another embodiment, the neutralizing antibody construct is directed against toxins produced by the bacteria. Examples of airborne bacterial pathogens include, for example, Neisseria meningitidis (meningitis), Klebsiella pneumoniae (pneumonia), Pseudomonas aeruginosa (pneumonia), Glandersinus meridian (pneumonia), Glandersinus rhinoplasia (pneumonia), Acinetobacter (pneumonia), Catarrococcus, Moraxella lacunata, Alcaligenes, Cardiobacterium, Haemophilus influenzae (influenzae). Streptococcus parainfluenza, Bordetella pertussis (pertussis), Bacillus tularensis (pneumonia / fever), Legionnaires' disease, Chlamydia psittacosis (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis (tuberculosis (TB)), Mycobacterium cansasi (TB), Mycobacterium avium (pneumonia), Nocardia asteroides (pneumonia), Bacillus anthrax (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacterium diphtheriae (diphtheria), Mycoplasma pneumoniae (pneumonia). ) is included.

[0098] rAAV may include genes encoding antibodies, particularly neutralizing antibodies against bacterial pathogens such as anthrax-causing substances, which are toxins produced by Bacillus anthrax. Neutralizing antibodies against the protective agent (PA), one of the three peptides that form the toxoid, have been described. The other two polypeptides consist of the lethal factor (LF) and the edema factor (EF). Anti-PA neutralizing antibodies have been described as effective for passive immunity against Bacillus anthrax. 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). Further anti-anthrax toxin neutralizing antibodies have been described and / or can be produced. Similarly, AAV-delivering antipathogen constructs, as described herein, can be produced using neutralizing antibodies against other bacteria and / or bacterial toxins.

[0099] Antibodies against infections may be caused by parasites or fungi, including, for example, Aspergillus, Abscidia corymbifera, Rhizopus, Mucor puruvea, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatichidis, Coccidioides imithis, Penicillium species, Micropolyspora pheni, Thermoactinomyces vulgaris, Alternaria alternata, Cladosporium species, Helmintosporium, and Stachybotrys species.

[0100] 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 are not limited to, but include, for example, alpha-synuclein, anti-vascular endothelial growth factor (VEGF) (anti-VEGF), anti-VEGFA, anti-PD-1, anti-PD-L-1, 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, and anti-beta cell synthesis. These may include mature antigens, anti-B lymphocyte stimulating factors, anti-CD20, anti-HER2, anti-granulocyte-macrophage colony stimulating factor, anti-oncostatin M (OSM), anti-lymphocyte activator gene 3 (LAG3) protein, anti-CCL20, anti-serum amyloid-P component (SAP), anti-prolyl hydroxylase inhibitors, 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-complementary protein C5, anti-CD11a, anti-CD3 receptor, anti-alpha-4 (α4) integrin, anti-RSV F protein, and anti-integrin α4β7. Further other pathogens and diseases will be apparent to those skilled in the art. Other suitable antibodies may include, for example, antibodies useful for treating Alzheimer's disease, particularly anti-beta-amyloid (e.g., crenezumab, solanezumab, adacnamab), anti-beta-amyloid fibrils, anti-beta-amyloid plaques, anti-tau, and bapineuzamab. Other suitable antibodies for treating various indications include, for example, those described in PCT / US2016 / 058968, filed on 27 October 2016 and published as WO2017 / 075119A1.

[0101] II. rAAV vector production For use in the production of AAV virus vectors (e.g., recombinant (r)AAV), the expression cassette can be supported on any suitable vector, such as a plasmid, to be delivered to the packaging host cell. Plasmids useful in this invention are, in particular, suitable for in vitro replication and packaging in prokaryotic cells, insect cells, and mammalian cells. They can be manipulated in this manner. Suitable transfection techniques and packaged host cells are known and / or can be easily designed by those skilled in the art.

[0102] Methods for generating and isolating AAV suitable for use as a vector are known in the art. Generally, 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, and Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J.Gene See Med.10:717–733 and the references cited below, each of which is incorporated herein by reference in its entirety. To package the transgene into a virion, the ITR is the only AAV component required in cis form within the same construct as the nucleic acid molecule containing the expression cassette. The cap and rep genes may be supplied trans form.

[0103] In one embodiment, the expression cassette described herein is engineered into a genetic element (e.g., a shuttle plasmid) that introduces an introduced gene construct sequence carried thereon into a packaging host cell in order to produce a viral vector. In one embodiment, the selected genetic element may be delivered to AAV packaging cells by any preferred method, including transfection, electroporation, liposome delivery, membrane fusion techniques, fast DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be produced. Alternatively, the expression cassette may be used to produce viral vectors other than AAV. Methods used to produce such constructs are known to technicians 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).

[0104] The term “AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid that lacks the desired genomic sequence it was packaged in. These may be referred to as “empty” capsids. Such capsids may contain no detectable genomic sequence at all for the expression cassette, or only a partially packaged genomic sequence that is insufficient to achieve gene product expression. These empty capsids are nonfunctional for introducing the gene of interest into host cells.

[0105] The recombinant adeno-associated viruses (AAVs) described herein can be produced using known techniques. See, for example, WO2003 / 042397;WO2005 / 033321, WO2006 / 110689;US7588772 B2. Such methods involve culturing host cells containing a nucleic acid sequence encoding the AAV capsid protein, a functional rep gene, an expression cassette consisting of at least the AAV reverse-terminal repeat (ITR) and a transgene, and sufficient helper function to allow the expression cassette to be packaged into the AAV capsid protein. Methods for producing the capsid, the coding sequence therefor, and methods for producing the rAAV viral vector are described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086(2003) and US 2013 / 0045186A1.

[0106] In one embodiment, a production cell culture useful for producing recombinant AAV is provided. Such a cell culture comprises a nucleic acid expressing the AAV capsid protein in a host cell, a nucleic acid molecule suitable for packaging into the AAV capsid, such as a vector genome containing AAV ITR, and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing the expression of the product in the host cell, as well as sufficient AAV rep and adenovirus helper functions to enable the packaging of the nucleic acid molecule into the recombinant AAV capsid. In one embodiment, the cell culture consists of mammalian cells (e.g., human embryonic kidney 293 cells in particular) or insect cells (e.g., baculovirus).

[0107] Optionally, the rep function is provided by an AAV other than the AAV that provides the capsid. For example, reps are not limited to these, but include 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 These may be 40, rep68 / 78, and rep40 / 52, or fragments thereof. Optionally, the rep and cap sequences reside on the same genetic element in the cell culture. A spacer may be present between the rep sequence and the cap gene. Either of these AAV or mutant AAV capsid sequences may be regulated by exogenous regulatory elements that direct their expression in the host cell.

[0108] In one embodiment, cells are produced in a suitable cell culture (e.g., HEK293) of cells. Methods for producing gene therapy vectors described herein include methods well known in the art, such as the production of plasmid DNA used for gene therapy vector production, vector production, and vector purification. In some embodiments, the gene therapy vector is an AAV vector, and the plasmids produced are an AAV cis plasmid encoding a vector genome containing the gene of interest, an AAV transplasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may include method steps such as initiating cell culture, passing cells through, seeding cells, transfection of cells with plasmid DNA, exchange to serum-free medium after transfection, and collection of vector-containing cells and culture medium. The collected vector-containing cells and culture medium are referred to herein as crude cell collections. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For reviews of these production systems, see, for example, Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for For large-scale recombinant adeno-associated virus production, see Human Gene Therapy 20:922–929, the contents of which are incorporated herein by reference in their entirety. Methods for manufacturing 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 entirety: 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.

[0109] Subsequently, the crude cell sample may undergo targeted method steps such as concentration of the vector sample, diafiltration of the vector sample, microfluidization of the vector sample, nuclease digestion of the vector sample, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration for preparing the bulk vector.

[0110] The vector drug product is purified by two steps of high-salt-concentration affinity chromatography followed by anion exchange resin chromatography to remove empty capsids. These methods are described in more detail in International Patent Publication WO2017 / 160360, which is incorporated herein by reference. The purification methods for AAV8 in Publication WO2017 / 100676, for rh10 in International Patent Publication WO2017 / 100704, and for AAV1 in International Patent Publication WO2017 / 100674 are all incorporated herein by reference.

[0111] To calculate the empty and complete particle content, the VP3 band volume for a selected sample (e.g., in the examples herein, the iodixanol gradient - purified preparation, where GC = number of particles) is plotted against the loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test sample peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. The ratio of particles to genome copies (pt / GC) is obtained by dividing Pt / mL by GC / mL. Pt / mL ~ GC / mL gives the empty pt / mL. The percentage of empty particles is obtained by dividing empty pt / mL by pt / mL and then multiplying by 100.

[0112] In general, assay methods for AAV vector particles with empty capsids and packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330 and Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsids, the method includes, for example, providing the treated AAV stock to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating three capsid proteins, such as a gradient gel containing 3-8% Tris-acetate in buffer; then running the gel until the sample material is separated; and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. The 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, which binds to the primary antibody and includes means for detecting binding to 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 a color change, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, a sample from a column fraction is collected and heated in an SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid protein is degraded 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, namely SYPRO ruby ​​or Coomassie staining, may be performed.In one embodiment, the concentration of the AAV vector genome (vg) in a column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, the sample is further diluted and amplified using a TaqMan® fluorescence-generating probe specific to the DNA sequence between primers and primers. The number of cycles required to reach a specified level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. A. A standard curve for Q-PCR reactions was created using plasmid DNA containing the same sequence as that contained in the AV vector. The vector genome titer was determined by normalizing the cycle threshold (Ct) value obtained from the sample against the Ct value of the plasmid standard curve. Digital PCR-based endpoint assays can also be used.

[0113] 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, treated with protease K, and subsequently inactivated by heat. Preferably, the sample is diluted with an amount of protease K buffer equal to the sample size. The protease K buffer may be concentrated more than 2-fold. 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 approximately 55°C for approximately 15 minutes, but may be carried out at a lower temperature (e.g., approximately 37°C to approximately 50°C) for a longer period (e.g., approximately 20 to approximately 30 minutes), or at a higher temperature (e.g., up to approximately 60°C) for a shorter period (e.g., approximately 5 to 10 minutes). Similarly, thermal inactivation is generally carried out at approximately 95°C for approximately 15 minutes, but may be performed at a lower temperature (e.g., approximately 70 to approximately 90°C) and for a longer period (e.g., approximately 20 to approximately 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

[0114] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for measuring single-stranded and self-complementary AAV vector genome titers by ddPCR has been described. See, for example, M. Lock et al, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131.Epub 2014 Feb 14.

[0115] In short, a method for separating rAAV particles having a packaged genome sequence from genome-deficient AAV intermediates comprises subjecting a suspension containing recombinant AAV virus particles and AAV capsid intermediates to high-performance liquid chromatography, where the AAV virus particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at high pH and subjected to a salt gradient while monitoring the eluate for ultraviolet absorbance at approximately 260 and 280. The pH may be adjusted depending on the selected AAV. See, for example, WO2017 / 160360 (AAV9), WO2017 / 100704 (AAVrh10), WO2017 / 100676 (e.g., AAV8), and WO2017 / 100674 (AAV1)), which are incorporated herein by reference. In this method, the complete AAV capsid is recovered from the fraction that elutes when the A260 / A280 ratio reaches the infection point. In one embodiment, for the affinity chromatography step, the diafiltration product may be applied to Capture Select® Poros-AAV2 / 9 affinity resin (Life Technologies) which efficiently captures AAV2 serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flows through the column, while AAV particles are efficiently captured.

[0116] III. Composition and Use This specification provides compositions comprising at least one rAAV stock (e.g., an rAAV stock or a variant rAAV stock) and an optional carrier, excipient, and / or preservative. An rAAV stock refers to multiple identical rAAV vectors, for example, in the amounts described below in the consideration of concentration and dose units.

[0117] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption retardants, 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 may 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, and vesicles may be used to introduce the compositions of the present invention into suitable host cells. In particular, rAAV vector delivery transgenes may be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0118] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically suitable pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate that is diluted for administration to a subject. In yet another embodiment, the composition may be lyophilized and reconstituted at the time of administration.

[0119] A suitable surfactant, or combination of surfactants, may be selected from among non-toxic nonionic surfactants. In one embodiment, for example, a primary hydroxyl-terminated bifunctional block copolymer surfactant such as Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400, may be selected. Other surfactants and other poloxamers, namely 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 hydroxysteart), 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 x100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x10 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.

[0120] The vector is administered in an amount sufficient to transfect cells, providing a sufficient level of gene transfer and expression to deliver a therapeutic effect without excessive adverse effects or with medically acceptable physiological effects, which can be determined by those skilled in the art. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery), lungs, heart, eyes, kidneys), orally, by inhalation, intranasal, intrathecal, intratracheal, intraarterial, direct delivery to the eye (optionally via intraocular delivery, subretinal injection, intraretinal injection, intravitreous, topical), intravenously, intramuscular, subcutaneous, intracutaneous, and direct delivery to other parental routes of administration. In one embodiment, the route of administration is subretinal or intravitreous injection. Routes of administration may be combined if desired.

[0121] The dosage of viral vectors depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore can vary among patients. For example, the therapeutically effective human dose of viral vectors is generally about 25 to 1000 microliters to 100 mL, or about 1 × 10⁶. 9 ~1 × 10 16 This is a range of solutions containing the concentration of the genomic viral vector. The dosage may be adjusted to balance the therapeutic benefits with any side effects. Such dosages may vary depending on the therapeutic application for which the recombinant vector is used. The expression level of the transgene can be monitored to determine the frequency of administration resulting in a viral vector, preferably an AAV vector containing a minigene. Optionally, drug regimens similar to those described for therapeutic purposes may be used for immunization using the compositions of the present invention.

[0122] The defective replication virus composition is formulated in dose units, and for human patients, it is approximately 1.0 × 10⁶ 9 GC~approx. 1.0×10 16 A quantity of replication-defective viruses within the GC range (to treat an average subject weighing 70 kg), including all integer or fractional quantities within that range, preferably 1.0 × 10⁻¹⁶. 12 GC~1.0×10 14It can be included within the range of GC. In one embodiment, the composition contains at least 1x10 per dose, including all integers or fractional amounts within the range. 9 、2x10 9 、3x10 9 、4x10 9 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、or 9x10 9 GC and is formulated to contain it. In another embodiment, the composition contains at least 1x10 per dose, including all integers or fractional amounts within the range. 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、or 9x10 10 GC and is formulated to contain it. In another embodiment, the composition contains at least 1x10 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 GC and is formulated to contain it. In another embodiment, the composition contains at least 1x10 per dose, including all integers or fractional amounts within the range. 12 、2x10 12 、3x10 12 、4x10 12 、5x10 12 、6x10 12 7x10 12 、8x10 12 、or 9x10 12 GC and is formulated to contain it. In another embodiment, the composition contains at least 1x10 per dose, including all integers or fractional amounts within the range. 13 、2x10 13 、3x10 13, 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 The composition is formulated to include GC. In another embodiment, the composition contains at least 1 x 10 per dose, including all integers or fractional quantities within the range. 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , or 9x10 14 The composition is formulated to include GC. In another embodiment, the composition contains at least 1 x 10 per dose, including all integers or fractional quantities within the range. 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15 It is formulated to contain GC. In one embodiment, for human application, the dose is 1 × 10 per dose, including all integers or fractional quantities within the range. 10 ~Approx. 1×10 12 It could be within the scope of garbage collection.

[0123] These above doses may be administered in higher volumes, including various volumes of carriers, excipients, or buffer formulations, or any number within that range, ranging from about 25 to about 1000 microliters, 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 another embodiment, the volume is about 50 μL. In yet another embodiment, the volume is about 75 μL. In yet another embodiment, the volume is about 100 μL. In yet another embodiment, the volume is about 125 μL. In yet another embodiment, the volume is about 150 μL. In yet another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In yet 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 approximately 325 μL. In yet another embodiment, the volume is approximately 350 μL. In yet another embodiment, the volume is approximately 375 μL. In yet another embodiment, the volume is approximately 400 μL. In yet another embodiment, the volume is approximately 450 μL. In yet another embodiment, the volume is approximately 500 μL. In yet another embodiment, the volume is approximately 550 μL. In one embodiment, the volume is approximately 600 μL. In another embodiment, the volume is approximately 650 μL. In yet another embodiment, the volume is approximately 700 μL. In yet another embodiment, the volume is approximately 700 to 1000 μL.

[0124] In certain embodiments, the dose is approximately 1 x 10 9 GC / g brain mass ~ approx. 1x10 12 The GC / g brain mass may be in the range. In certain embodiments, the dose is approximately 3 x 10⁻⁶ 10 GC / g brain mass ~ approx. 3x10 11 The GC / g brain mass may be in the range. In certain embodiments, the dose is approximately 5 x 10 10 GC / g brain mass ~ approx. 1.85x10 11 It could be within the range of GC / g brain mass.

[0125] In another embodiment, an aqueous suspension suitable for administration to a subject is provided. In one embodiment, the suspension is an aqueous suspension of recombinant adeno-associated virus (rAAV) as described herein, useful as a therapeutic agent for the treatment or prevention of eye diseases, and approximately 1 x 10 per eye. 9 Virus particles ~ approximately 1 x 10 13 It contains GC or virus particles. In one embodiment, the suspension is suitable for subretinal or intravitreous injection.

[0126] In one embodiment, the virus construct is at least about 1 × 10 9 GC~approx. 1×10 15 GC, or approximately 1 × 10⁻⁶ 11 GC~5×10 13 It can be delivered in GC doses. Suitable volumes for delivering these doses and concentrations can be determined by those skilled in the art. For example, volumes of about 1 μL to 150 mL may be selected, but higher volumes may be selected for adults. Typically, for neonates and infants, appropriate volumes may be 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, volumes of about 0.5 mL to about 20 mL may be selected. For children, volumes up to about 30 mL may be selected. For preteens and teenagers, volumes up to about 50 mL may be selected. In yet another embodiment, the patient may receive intrathecal administration in volumes 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. Doses may be adjusted to balance the therapeutic benefits with any side effects, and such dosages may vary depending on the therapeutic use for which the recombinant vector is utilized.

[0127] The recombinant vectors described above can be delivered to host cells according to the published methods. Preferably, rAAV suspended on a physiologically compatible carrier can be administered to human or non-human mammalian patients. In certain embodiments, for administration to human patients, rAAV is preferably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt, or a mixture of salts. Preferably, the formulation is adjusted to a physiologically acceptable pH range, for example, pH 6–9, or pH 6.5–7.5, pH 7.0–7.7, or pH 7.2–7.8. Since the pH of cerebrospinal fluid is approximately 7.28–7.32, a pH within this range may be desirable for intrathecal delivery, and a pH of approximately 6.8–7.2 may be desirable for intravenous, subretinal, or intravitreous delivery. However, other pH ranges, and sub-ranges of these, are available and may be selected for other delivery routes.

[0128] In another embodiment, the composition comprises a carrier, a diluent, an excipient, and / or an adjuvant. A suitable carrier can be readily selected by those skilled in the art, taking into account the indication to which the introduced virus is directed. For example, one suitable carrier comprises saline and 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 tube but do not interfere with rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, can be selected from non-toxic nonionic surfactants. In one embodiment, for example, Pluronic®, also known as poloxamer 188, has a neutral pH and an average molecular weight of 8400. A bifunctional block copolymer surfactant with a primary hydroxyl group terminus, such as )F68[BASF], is selected. Other surfactants and other poloxamers, namely 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 hydroxysteart), 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 a three-digit number, where the first two digits x100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. Surfactants may be present in amounts of up to approximately 0.0005% to approximately 0.001% of the suspension. In one embodiment, the formulation may contain a buffered saline solution containing, for example, one or more of the following in water: 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. Preferably, for intrathecal delivery, the molar osmotic pressure concentration is within a range compatible with cerebrospinal fluid (e.g., approximately 275 to approximately 290); see, for example, emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, commercially available diluents may be used as suspending agents or in combination with other suspending agents and any other excipients; see, for example, Elliotts B® solution [Lukare Medical]. In other embodiments, the formulation may contain one or more osmotic enhancers.Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0129] Optionally, the compositions of the present invention may include other conventional pharmaceutical components, such as preservatives or chemical stabilizers, in addition to rAAV and the carrier(s). 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.

[0130] The compositions according to the present invention may comprise a pharmaceutically acceptable carrier as defined above. Preferably, the compositions described herein comprise one or more effective amounts of AAV suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed for delivery to a target via infusion, permeation pump, intrathecal catheter, or by another device or route. In one embodiment, the composition is formulated for intrathecal delivery.

[0131] As used herein, the terms “intrathecal delivery” or “intrathecal administration” refer to the route of drug administration by injection into the spinal canal, more specifically, by injection into the subarachnoid space to reach the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including lateral ventricles (ICV)), suboccipital / cisternal, and / or C1-2 puncture. For example, a material may be introduced by lumbar puncture to diffuse across the subarachnoid space. In another embodiment, the injection may be into the cisterna magna.

[0132] As used herein, the terms “intracisternal delivery” or “intracisternal administration” refer to a direct route of drug administration into the cerebrospinal fluid of the cisterna magna, cerebellum, medulla oblongata, more specifically by suboccipital puncture, or This refers to a route of drug administration via a permanently placed tube, involving direct injection into the cisterna.

[0133] In one embodiment, the vectors provided herein may be administered intrathecally via methods and / or devices. See, for example, WO2017 / 181113, which is incorporated herein by reference. Alternatively, other devices and methods may be selected. The method includes the steps of advancing a spinal needle into the cisterna magna of a patient, connecting a flexible tube to the proximal hub of the spinal needle and connecting the outlet port of a valve to the proximal end of the flexible tube, and after the advancement and connection steps, making the tube self-primed with the patient's cerebrospinal fluid, connecting a first container containing a certain amount of isotonic solution to the flush inlet port of the valve, and then connecting a second container containing a certain amount of 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 valve outlet port, and the pharmaceutical composition is injected into the patient through the spinal needle. After the injection of the pharmaceutical composition, a passage for fluid flow is opened through the flush inlet port and the valve outlet port, and an isotonic solution is injected into the spinal needle to flush the pharmaceutical composition into the patient.

[0134] This method and this device may, optionally, be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.

[0135] Please note that the terms "a" or "an" refer to one or more. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0136] The terms “comprise,” “comprises,” and “comprising” should be interpreted comprehensively, not exclusively. The terms “consist,” “consisting,” and their variations should be interpreted exclusively, not comprehensively. While various embodiments herein are indicated using the term “comprising,” in other contexts, the relevant embodiments are also intended to be interpreted and described using the terms “consisting of” or “consisting essentially of.”

[0137] As used herein, the term “about” means a variability of 10% (±10%) from the given reference, unless otherwise specified.

[0138] As used herein, “disease,” “disorder,” and “condition” are used interchangeably to describe an abnormal condition in the subject.

[0139] Unless otherwise defined herein, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art and by reference to published documents that provide general guidance to those skilled in the art for the many terms used herein.

[0140] The term “expression” is used herein in its broadest sense and includes the production of RNA or RNA and proteins. With respect to RNA, the terms “expression” or “translation” are used in particular with respect to the production of peptides or proteins. Expression can be transient or stable.

[0141] As used herein, the term “NAb titer” is a measure of how much neutralizing antibody (e.g., anti-AAV Nab) is produced that neutralizes the physiological effects of its targeted epitope (e.g., AAV). Anti-AAV NAb titer can be measured as described, for example, 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.

[0142] As used herein, “expression cassette” refers to a nucleic acid molecule containing a coding sequence, a promoter, and other regulatory sequences for them, which may be delivered to a host cell by a genetic element (e.g., a plasmid) and packaged into a viral vector capsid (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector includes the coding sequence of the gene product described herein, adjacent to the packaging signal of the viral genome, and other expression regulatory sequences such as those described herein.

[0143] The abbreviation "sc" indicates self-complementary nature. "Self-complementary AAV" refers to a construct in which the coding region of the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of 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," Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference.

[0144] As used herein, the term “operably linked” refers to both an expression regulatory sequence that is continuous with the gene of interest and an expression regulatory sequence that acts in trans with the gene of interest or at a distance that controls the gene of interest.

[0145] When used in reference to proteins or nucleic acids, the term “heterogeneous” indicates that a protein or nucleic acid contains two or more sequences or subsequences that are not found in the same relationships to each other as they are in nature. For example, nucleic acids having two or more sequences from unrelated genes arranged to create a novel functional nucleic acid are typically produced by recombination. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to lead to the expression of coding sequences from different genes. Thus, with respect to the coding sequences, the promoter is heterogeneous.

[0146] A “replication-deficient virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing the gene of interest is packaged within a viral capsid or envelope, and any viral genome sequence packaged within the viral capsid or envelope is replication-deficient, i.e., they cannot produce progeny but can 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 is adjacent to the signals required for amplification and packaging of the artificial genome). These genes can be engineered to be "gutless," containing only the target transgene, and can be supplied during production. Therefore, they are considered safe for use in gene therapy because replication and infection by progeny virions cannot occur without the presence of the viral enzymes required for replication.

[0147] In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endo- and exonucleases may be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases may be selected to degrade single-stranded DNA and / or double-stranded DNA, and RNA. Such steps may involve a single nuclease or a mixture of nucleases directed to different targets, and may be endonucleases or exonucleases.

[0148] The term "nuclease resistance" indicates that the AAV capsid is fully constructed around an expression cassette designed to deliver the transgene to the host cell, protecting these packaged genomic sequences from degradation (digestion) during the nuclease incubation step, which is designed to remove any contaminating nucleic acids that may be present in the production process.

[0149] In the context of this invention, the term "translation" refers to a process in ribosomes in which an mRNA chain controls the assembly of amino acid sequences to produce a protein or peptide.

[0150] As used throughout this specification and the claims, the terms “comprising” and “including” include other components, elements, integers, steps, etc. Conversely, the term “consisting” and its variations exclude other components, elements, integers, steps, etc.

[0151] As mentioned above, unless otherwise specified, the term "approximately" means a variation of ±10% when used to adjust a number.

[0152] The following examples are illustrative and not intended to limit the invention. [Examples]

[0153] The following examples report extensive deamidation of AAV8 and additional diverse AAV serotypes, supported by evidence from structural, biochemical, and mass spectrometry approaches. The degree of deamidation at each site was dependent on the vector's age and multiple primary sequences and 3D structure factors, but largely independent of vector recovery and purification conditions. We demonstrate the potential of deamidation to affect vector transduction activity and correlate early-point losses in vector activity at some AAV8 asparagines with rapidly progressing spontaneous deamidation. We explore mutation strategies to stabilize side-chain amides, improve vector transduction, and reduce lot-to-lot molecular variability, a key concern in biopharmaceutical manufacturing. This study reveals previously unknown aspects of AAV capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

[0154] Example 1 characterizes post-translational modifications to AAV8 vector capsids using one-dimensional and two-dimensional gel electrophoresis, mass spectrometry, and novel structural modeling. Following the identification of multiple putative deamidation sites on the capsid surface, the impact on capsid structure and function is evaluated 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 and confirm that AAV capsid deamidation is not serotype specific. Examples 2-5 demonstrate deamidation of different AAVs. .

[0155] 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, the AAV vector was first denatured at 80°C for 20 minutes in the presence of lithium dodecyl sulfate and a reducing agent. Next, it was electrophoresed on a 4-12% Bis-Tris gel at 200V for 90 minutes and stained with Coomassie blue. For the data in Figures 1A-1D, Kendrick Laboratories, Inc. (Madison, WI) performed 2D gel electrophoresis. Subsequent experiments were performed in-house using 35 mM SDS-PAGE. In 150 μL of phosphate-buffered saline (PBS) containing NaCl and 1 mM MgCl2, 3 x 10 11The GC AAV vector and 500U turbonuclease marker (Accelagen, San Diego, CA) were combined and incubated at 37°C for 10 minutes. Next, nine times the volume of anhydrous ethanol was added, the sample was vortexed, and incubated at -80°C for at least 2 hours, followed by incubation on ice for 5 minutes. Nine times the volume of es was added, and the mixture was centrifuged at maximum speed at 15°C for 30 minutes. The supernatant was decanted, the pellet was 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 amphoteric electrolyte, ThermoFisher ZM0023 added to ddH2O on the day] and gently incubated at room temperature. After 30 minutes, the sample tubes were flicked to mix, 1 μg of chicken con albumin marker (Sigma Aldrich, St. Louis, MO) was added, and the samples were incubated at 37°C for 30 minutes, flicked to mix after 15 minutes. Next, the samples were transferred to 50°C for 15–20 minutes, vortexed, incubated at 95°C for 2.5 minutes, cooled, centrifuged at maximum speed for 1 minute, and briefly vortexed. Then, 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 the aforementioned 0.05% amphoteric electrolyte added to ddH2O on the same day) and incubated at room temperature for 10 minutes. The mixtures were then applied to pH 6–10 fixed 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. Strips were electrophoresed at 100–1,000 V for 120 minutes, 1,000–2,000 V for 120 minutes, and 2,000 V for 120 minutes, with limitations of 0.1 W and 0.05 mA per strip. 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 compared to internal control protein turbonuclease (Accelagen, 27 kDa) and chicken egg white conalbumin (Sigma Aldrich, 76 kDa, pI 6.0–6.6).

[0156] 2. Vector Production Vector cores from the University of Pennsylvania were used to prepare recombinant AAV vectors for 1D and 2D gel electrophoresis and mass spectrometry experiments, and were purified using cesium chloride or iodixanol gradients as described above. (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 vectors were produced as follows: HEK293 cells were grown in 10 36-layer hyperstack vessels (Corning) and co-transfected with a mixture of vector genome plasmid (pAAV-LSP-IVS2.hFIXco-WPRE-bGH), transplasmids containing AAV2 rep and AAV8 cap genes, and adenovirus helper plasmids. PEIpro (PolyPlus) was used as the transfection reagent. Five days after transfection, the supernatant was collected and S The solution was clarified through an artoguard PES Midicap filter (Sartorious Stedim), treated with benzonase (Millipore), and then diluted to 0.6 M with salt. The clarified bulk recovered material was concentrated 10-fold by tangential flow filtration (TFF) and then diafiltered into 4 volumes of affinity column packing buffer. The vector was captured on a POROS CaptureSelect (ThermoFisher) affinity column, and the vector peak was eluted directly into neutralizing buffer at low pH. The neutralized eluate was diluted in high pH binding buffer and loaded onto an anion exchange polishing column (Cimultus QA-8; Bia Separations) to concentrate the preparation for genome-containing (complete) particles. Complete 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 to formulation buffer (PBS + 0.001% Pluronic F-68).

[0157] 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 delta-F6 Ad helper plasmid in 90 μL serum-free medium) was mixed in 5.6 μL of 1 mg / mL polyethyleneimine solution and 90 μL of serum-free medium, incubated at room temperature for 15 minutes, added to cells, and then 0.8 mL of fresh serum-free medium was added. The following day, 0.5 mL of the top medium was replaced with whole serum medium. Three days after transfection, the vector was recovered by three freeze / thaw cycles, then centrifuged to remove cell debris, and the supernatant was collected. The cis plasmid contains a transgene cassette encoding a firefly cyferase transgene under the control of a chicken beta-actin (CB7) promoter with a promega chimeric intron and rabbit beta-globin (RBG) polyadenylation signal. The transplasmid encodes the wtAAV8 cap gene, and the Quikchange Lightning Mutagenesis Kit (Agilent Technologies, Wilmington, DE) was used to generate mutant AAV8 cap variants. The vector was titrated as described above (Lock M, et al. Hum Gene Ther 2010;21(10):1259-71).

[0158] For time-course vector generation experiments, vectors were generated by medium-scale triple transfection of HEK293 cells in 15 cm tissue culture dishes. Per plate, 36 μL of 1 mg / mL polyethyleneimine solution in 2 mL of serum-free medium was mixed with plasmid DNA (0.6 μg cis plasmid, 5.8 μg trans plasmid, 11.6 μg delta-F6 Ad-helper plasmid), incubated at room temperature for 15 minutes, and added to cells at approximately 60% concentration on plates refreshed with 14 mL of serum-free medium. The following day, 8 mL of top medium was replaced with fresh, complete serum medium. All top medium was collected, cells were scraped from the dish, and the vector was recovered by freezing 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 for mass spectrometry was purified and concentrated by adding benzonase, 1 M Tris pH 7.5, and 5 M NaCl to the clarified lysate to final concentrations 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 neutralizing buffer at low pH. The fractions were analyzed by absorption at 280 nm, and the most concentrated fraction was subjected to mass spectrometry.

[0159] For in vivo experiments, a vector was produced using either the aforementioned wtAAV8 capsid or one of the six deamidation mutants, and the transgene cassette contained the CB7 promoter, PI intron, firefly luciferase transgene, and RBG polyadenylation signal (Lock M, et al. Hum Gene Ther 2010;21(10):1259-71).

[0160] 3. Mass Spectrometry Execution / Digest / Analysis Materials: Ammonium bicarbonate, DTT, iodoacetamide (IAM), and 18O concentrated water (97.1% purity) were purchased from Sigma (St. Louis, MO), and acetonitrile, formic acid, trifluoroacetic acid (TFA), 8M guanidine hydrochloride (GndHCl), and trypsin were purchased from Thermo Fischer Scientific (Rockford, IL).

[0161] Trypsin digestion: Stock solutions of 1M DTT and 1.0M iodoacetamide were prepared. Capsid proteins were denatured and reduced at 90°C for 10 minutes in the presence of 10 mM DTT and 2 M GndHCl. The samples were cooled to room temperature and then alkylated at room temperature for 30 minutes in the dark using 30 mM IAM. The alkylation reaction was quenched by adding 1 mL of DTT. 20 mM ammonium bicarbonate (pH 7.5-8) was added to the denatured protein solution to dilute the final GndHCl concentration to 200 mM. Trypsin solution was added to achieve a trypsin-to-protein ratio of 1:20 and incubated overnight at 37°C. After digestion, TFA was added to a final concentration of 0.5% to quench the digestion reaction.

[0162] In the 18O-water experiment, the capsid sample was first buffered with 100 mM ammonium bicarbonate prepared in 18O-water using a Zeba spin desalination column (Thermo Scientific, Rockford, IL). Two buffer exchanges were performed to completely remove water from the sample. Stock solutions of 1 M DTT and 1 M IAM were prepared in 18O-water. The denaturation, alkylation, and digestion steps were followed using the 18O-water reagent and buffer as described above.

[0163] Liquid chromatography-tandem mass spectrometry (LCM) was performed online using a Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific) coupled with a Q Exactive HF equipped with an Acclaim PepMap column (15 cm length, 300 μm inner diameter) and a NanoFlex source (Thermo Fisher Scientific). The column temperature was maintained at 35°C during the online analysis. Peptides were separated by a gradient of mobile phase A (MilliQ water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid). The gradient was run from 4%B to 6%B over 15 minutes, to 10%B over 25 minutes (total 40 minutes), and then to 30%B over 46 minutes (total 86 minutes). Samples were loaded directly onto the column. The column size was 75 cm × 15 μm ID and packed with 2 microns of C18 medium (Acclaim PepMap). The total time for each liquid chromatography-tandem mass spectrometry run, including loading, introduction, and washing steps, was approximately 2 hours.

[0164] Mass spectrometry data was acquired using a data-dependent top-20 method in a Q Exactive HF mass spectrometer, and the most abundant unsequenced precursor ions were dynamically selected from a survey scan (200–2000 m / z). Sequencing was performed via higher-energy collision dissociation fragmentation at a target value of 1e5 ions determined by predictive auto-gain control, and precursor isolation was performed with a 4 m / z window. The survey scan was acquired at 200 m / z with a resolution of 120,000. The HCD spectral resolution was set to 30,000 at m / z200 with a maximum ion implantation time of 50 ms and a normalized collision energy of 30. The RF level of the S lens was set to 50, which optimally transfers the m / z region occupied by peptides from the digest. Precursor ions with a single, unassigned, or more than 6 charge states were excluded from fragmentation selection.

[0165] Data processing: BioPharma Finder 1.0 software (Thermo All acquired data were analyzed using Fischer Scientific. For peptide mapping, carbamide methylation was set as the fixed modification, and oxidation, deamidation, and phosphorylation were set as variable modifications, and searches were performed using the single-entry protein FASTA database. Tandem mass spectrometry spectra were used with a mass precision of 10 ppm, high protease specificity, and a confidence level of 0.8. Since deamidation adds the mass of the intact molecule + 0.984 Da (mass difference of -OH and -NH2 groups), mass spectrometric identification of deamidated peptides is relatively straightforward. The percentage of deamidation of a particular peptide was determined by dividing the mass area of ​​the deamidated peptide by the sum of the areas of the deamidated and native peptides. Given the number of possible deamidation sites, isotonic species deamidated at different sites may co-migrate in a single peak. Therefore, multiple deamidation sites can be identified or distinguished using fragment ions derived from peptides with multiple potential deamidation sites. In these cases, the relative intensities within the observed isotopic patterns can be used to specifically determine the relative abundances of different deamidated peptide isomers. This method assumes that the fragmentation efficiency is the same for all isomer species and that they are independent at the deamidation site. This approach allows for the definition of specific sites involved in deamidation and potential combinations involved in deamidation.

[0166] Secondary Data Processing: Secondary analysis of raw mass spectrometry 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 spectrometry. Data refinement of raw data files was performed using a precursor m / z tolerance of ≤10 ppm and precursor charge state parameters of a minimum of 2 and a maximum of 4. Novel 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 digestive enzyme was set as trypsin, with variable modifications being oxidation, phosphorylation, and deamidation, and the fixed modification being cysteine ​​carbamide methylation.

[0167] 4. Structural analysis of AAV capsids Atomic coordinates, structure factors, and associated capsid models of AAV8 were obtained from the RCSB Protein Data Bank (PDB number: 3RA8). Structural 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 the electron density of isoaspartic acid in the AAV8 capsid, unbiased by the expected primary sequence of AAV8 VP3. Using the obtained structure, the four asparagines in the AAV8 VP3 primary sequence, with N+1 glycine as isoaspartic acid, were modeled. The AAV8 capsid structure was then purified using crystallography and NMR System (CNS) software by strictly forcing an icosahedral amorphous matrix using a standard purification protocol (Brunger AT, et al. Acta Crystallogr). D Biol Crystallogr 1998;54(Pt 5):905-21). A structural model of isoaspartic acid was obtained from the HIC-UP database, followed by the generation of a molecular dictionary in PRODRG for structural purification (Kleywegt GJ Acta Crystallogr D Biol Crystallogr 2007;63(Pt 1):94-100). Next, the average electron density map of the AAV8 capsid (also in the CNS) was calculated and visualized using COOT software, and then minor adjustments were made to the resulting model to fit the modeled isoaspartic acid residues to 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 include N+1 glycine. N512 in the AAV9 VP3 primary sequence was further modeled (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). Multiple structures of previously identified deamidated proteins were obtained to compare the electron density maps of deamidated isoaspartate residues with the modeled isoaspartate residues of AAV8 and AAV9 (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).

[0168] The temperature coefficient of the deamidated residue was determined by averaging the temperature coefficients of each atom in each asparagine residue reported in the atomic coordinates of the AAV8 or AAV9 crystal structure (PDB numbers: 3RA8, 3UX1).

[0169] 5.Animal research The University of Pennsylvania's Institutional Animal Care and Use Committee approved all animal procedures. To evaluate vector performance, 8-week-old C57BL / 6 mice were intravenously injected with 100 μL of 3e10 GC wtAAV8 or capsid mutant vector via tail vein infusion. 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 of luciferin substrate (Perkin Elmer, Waltham, MA). Mice were imaged 5 minutes after luciferin administration via the IVIS Xenogen in vivo imaging system. Signals in the region of interest were quantified using LivingImage 3.0 software. Measurements were taken on days 7 and 14.

[0170] 6. Evaluation of mutant vector titer and in vitro transduction efficiency The vector titer was determined by qPCR of the DNAseI-resistant genome. The qPCR primers anneal to the polyadenylation sequence of the packaged transgene. For in vitro evaluation of vector transduction efficiency by luciferase expression, 0.9e5 Huh7 cells / well were seeded in a black-walled 96-well plate with complete DMEM (10% fetal bovine serum, 1% penicillin / streptomycin). The next 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 three-fold dilution series. After 48 h, luciferin (Promega, Madison, WI) in complete medium at 0.3 μg / μL was prepared and added to the transduced cells in a 50-μL volume. The results were read on a Biotek Clarity luminometer. Luciferase activity / GC added to the target cells was found to be constant over a wide range of GC but could saturate at high MOI. Therefore, the linearity of the dilution series data (luminescence units vs. GC) was examined, the highest points where saturation was evident were excluded, and the average luciferase / GC was calculated for values within the linear range of each assay for each variant. This yielded the transduction efficiency values. The data were normalized to simplify comparison by setting the value of the Wt control to 1.

[0171] 7. In Vivo Distribution DNA was extracted from liver samples using the QIAamp DNA Mini Kit (Qiagen, Hilden, German y), and then the DNA of vector GC was analyzed by real-time PCR as described above using a primer / probe set designed for the RBG polyadenylation signal of the transgene cassette (Chen SJ, et al. Hum Gene Ther Clin Dev 2013;24(4):154-60). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0172] B. Results AAV8 exhibits substantial charge heterogeneity in its capsid protein.

[0173] To qualitatively assess the presence of post-translational modifications on the AAV8 vector capsid that may affect vector performance, the AAV8 total capsid protein, purified by an iodixanol gradient, was analyzed by both 1D and 2D gel electrophoresis. On a 1D reduced sodium dodecyl sulfate SDS gel, VP1, VP2, and VP3 were degraded as single bands with 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 degraded as a series of distinct spots with different isoelectric points (pI) in the pH 6.3 to >7.0 range, dependent on the VP isoform (Figure 1D). The individual spots of each VP, when measured as migrations compared to the internal isoelectric point standard of carbonic anhydrase isoform, were separated at discrete intervals of 0.1 pI units, suggesting single-residue charge changes. The presence of these isoforms suggests that each VP has the potential to undergo numerous modifications, thereby resulting in different migrations under isoelectric focusing.

[0174] Deamidation (Figure 1A), in which a portion of the side-chain amide group (typically asparagine) is converted to a carboxylic acid, is a common source of charge isomerism in protein preparations. To determine if deamidation is responsible for different 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 main spots of VP1, VP2, and VP3 are shifted by one spot position (0.1 pH units) compared to the equivalent spot in wild-type (wt) AAV8 (Figures 1E-1G). The magnitude of this shift corresponds to the observed spacing between wt VP charge isoforms. Thus, the 2D gel pattern of the AAV capsid protein is consistent with multi-site deamidation.

[0175] Spontaneous deamidation occurs in AAV8 vector capsids. To identify the modifications responsible for the distinct spotting patterns of each capsid protein, a panel of AAV8 vectors was analyzed by mass spectrometry. The coverage of the AAV8 capsid proteins exceeded 95% of the total VP1 sequence on average (data not shown). Mass spectrometry detected extensive deamidation of subsets of asparagine and glutamine residues, showing an increase of approximately 1 Da in the observed mass of individual peptides compared to predicted values ​​based on the DNA-encoded sequence. This deamidation pattern was observed in all preparations of the AAV8 vector (Figures 2A–2D).

[0176] To assess the overall heterogeneity of deamidation between commonly used purification methods and to investigate 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 cis plasmids (producing only empty capsids) were also evaluated and purified by an iodixanol gradient.

[0177] Extensive deamidation was present across asparagine and glutamine residues of the AAV8 capsid, ranging from undetectable levels to over 99% of individual amino acids that were deamidated (Figure 2E). The highest level of deamidation (over 75%) occurred at asparagine residues where the N+1 residue was glycine (i.e., the NG pair) (Table 1). Lower levels of deamidation (i.e., up to 17%) were detected at additional asparagine residues where N+1 was not glycine. The mean deamidation of asparagine was nearly consistent across preparations. Deamidation was also detected at glutamine residues, but at a lower frequency than at asparagine, with the highest percentage observed being less than 2% at Q467 (Figure 7). This observation was inconsistent across preparations (data not shown). The largest 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 variability observed in deamidation between vector preparations 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 6]

[0178] Next, a series of experiments were performed to determine whether the sample treatment contributed to the observed level of deamidation in AAV8. Extreme temperatures (70°C for 7 days) or pH (pH 2 or pH 10 for 7 days) did not significantly induce additional deamidation in the AAV8 capsid. (Figures 4A and 4B). Given this resistance, we consider that the observed deamidation was unlikely to have occurred only during the purification stage, and was shorter and relatively milder than that. We attempted to perform mass spectrometry on the unpurified vector to determine the degree of deamidation before and after purification, but was unsuccessful. Similarly, the heavy water control indicates that processing specific to the mass spectrometry workflow does not contribute to additional deamidation events (Figure 4C).

[0179] To validate the mass spectrometry workflow, we investigated two recombinant proteins previously evaluated for deamidation, and our findings (Figures 5A and 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]. Furthermore, a secondary laboratory was hired to evaluate the raw data from AAV8. This independent analysis identified the same sites as the deamidated sites, and the variation in the degree of correction at each site due to software variability in peak detection and area calculation was minimal (Figure 6).

[0180] Structural morphology, temperature factors, and the identity of the N+1 amino acid contribute to the deamidation frequency. The structure of AAV8 has been analyzed and published (PDB identifier: 2QA0) (Nam HJ, et al. J Virol 2011;85(22):11791-99). Next, the AAV8 capsid structure was investigated for evidence of favorable conditions for non-enzyme deamidation, and the deamidation rate was correlated with established structural features (Nam HJ, et al. J Virol 2007;81(22):12260-71). Factors influencing asparagine deamidation are better characterized in the literature, and since asparagine deamidation events are far more common than glutamine deamidation events, the study focused solely on asparagine residues (Robinson, NE, and Robinson, AB (2001). Molecular clocks.Proc Natl Acad Sci USA 98:944-949). Also, the temperature (or B) factor of each of these residues was determined from the AAV8 crystal structure, where the temperature factor is a measure of the displacement of the atom from its mean position, and a higher value indicates a greater displacement, higher thermal vibration, and therefore increased flexibility (Parthasarathy). S and Murphy MR. Protein Science: A Publication of the Protein Society 1997;6:2561-7). The majority of the target asparagine is located within or near the surface-exposed HVR (Table 1), which is structurally favorable for deamidation and provides an environment for exposure to the solvent (Govindasamy L, et al. J Virol 2013;87(20):11187-99). We found that residues located in these flexible loop regions are deamidated more frequently on average than residues in less flexible regions such as the beta chain and alpha helix. For example, the NG residue at position N263 is part of HVR I, has a high-temperature factor, and was deamidated more than 98% on average (Figure 7A and Figure 6, Table 1). N514, which was deamidated for approximately 85% of the time (Figures 3 and 6, Table 1), is also present in HVR (HVR V) with N+1 glycine; however, its local temperature factor is relatively low compared to N263 because it interacts with residues on other VP monomers in the triple axis. Unfavorable +1 residues and lower local temperature factors correlate with lower deamidation, even in HVR residues. For example, N517 was deamidated for only 4% on average (Table 1) This residue has a temperature factor equivalent to that of highly deamidated N514, but its N+1 residue is serine, which reduces the possibility of sterically hindered deamidation. 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.

[0181] To test the role of the +1 residue in asparagine deamidation, mutant vectors were generated in which the AAV8 NG site was individually mutated to either alanine or serine at the +1 position. Model peptide studies indicate that the NG peptide deamidates with a half-life of about 1 day, whereas the NA or NS peptides typically deamidate 25 or 16 times slower, respectively (Robinson NE and Robinson AB. Proc Natl Acad Sci USA. 2001;98(8):4367-72). Mass spectrometry of the vector variants confirmed the central role of the +1 site in determining the extent of vector deamidation. The NG site in this set (with >80% deamidation in the wt) showed selective stabilization of the adjacent asparagine when the +1 site was changed to alanine (<5% deamidation) or serine (<14% deamidation) (Table 2).

Table 7

[0182] Residues located in regions of low local flexibility in the at least partially buried, not readily solvent-exposed, and / or intact fully assembled AAV8 capsid had a lower frequency of deamidation compared to those in more favorable environments (Table 1). Nevertheless, some of the residues in unfavorable states were deamidated. For example, N630, which is at least partially buried, still had a detectable level of deamidation. For this residue, the presence of phenylalanine as the N+1 residue suggests that this region is an AAV8 Suggests a possible novel site for non-enzymatic autoproteolytic cleavage in the VP3 protein.

[0183] AAv8 VP3 structural modeling confirms deamidation events 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 is therefore insufficient to directly distinguish between asparagine, aspartic acid, and isoaspartic acid residues. Other aspects of the structures 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 the following two assumptions: 1) The main product of the 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), and 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 allows the beta carbon from the R group of isoaspartic acid to be incorporated into the backbone of the AAV8 VP3 capsid protein backbone after the degradation of the succinimidyl intermediate during the deamidation reaction. It is created when it is lost.

[0184] First, the AAV8 structure itself was modified to generate an AAV8 capsid electron density unbiased by the known AAV8 VP3 sequence. Next, the purified AAV8 crystal structure was examined for evidence of deamidation based on the presence of shorter R groups associated with isoaspartic acid (Figures 3A-3E). The electron density map confirmed shorter R groups at highly deamidated N+1 glycine residues at positions 263 (Figure 3C), 385 (not shown), 514 (Figure 3D), and 540 (Figure 3E) compared with asparagine at position 410, which did not have 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 at these sites, which show more than 75% deamidation. The obtained isoaspartic acid model was equivalent to the isoaspartic acid residue observed in the crystal structures of other known deamidated proteins, thus validating the analysis of AAV8 (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). This structural analysis serves as an independent confirmation of the deamidation phenomenon observed when analyzing the AAV8 capsid via mass spectrometry.

[0185] Deamidation of AAV capsids is not serotype-specific. As evidence of capsid deamidation, serotypes beyond AAV8 were investigated. AAV9 vector preparations were examined using 2D gel electrophoresis (Figure 11A) and mass spectrometry (Figure 11B), including controls for potential vector processing effects (Figures 11D–11F). The pattern and degree of AAV9 deamidation were similar to those of AAV8. All four AAV9 NG sites were deamidated by more than 85%, thirteen non-NG sites were deamidated to less extent, and some sites showed high lot-to-lot variability in deamidation percentage. Next, a structural analysis workflow was applied to refit existing AAV9 crystal data (Figure 11C, Table 3). Similar to AAV8, isoaspartic acid is more suitable for 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 exhibited similar patterns and degrees of deamidation, indicating that this modification is widespread in clinically relevant AAV vectors and is determined by similar fundamental primary sequence and structural factors. [Table 8] [Table 9]

[0186] Deamidation events can affect capsid assembly and transduction efficiency. One approach to test the functional effects of deamidation is to replace asparagine with aspartate through genetic mutation. Small-scale triple transfection of 293 cells was used to evaluate the luciferase levels of each deamidated AAV8 asparagine. Aspartate mutant vectors encoding the vector were generated, and the vector titer was measured by qPCR of DNAseI-resistant genomic 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 with low overall deamidation in the wt vector. Next, the mutation panel was evaluated for in vitro transduction efficiency of human liver-derived Huh7 cells (Figure 8B). Several mutants showed reduced transduction efficiency, with positions N57, N94, N263, N305, Q467, N479, and N653 showing transduction loss of more than 10-fold. A similar number of susceptible sites were observed for AAV9 (Figures 11G and 11H). Typically, since only a subset of residues at a given site are endogenously deamidated, this approach may overestimate the loss of function in proteins such as capsids, where the functional unit is a homomeric aggregate, and 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 manufacturing or mutation stabilization. To place this loss-of-function mutagenesis data in the appropriate context, functional data from a population of endogenously deamidated vectors would be necessary.

[0187] Loss of vector activity over time correlates with progressive deamidation. Given the clearly short half-life of NG deamidation, we inferred that vector samples differing in age by only one day would exhibit different deamidation profiles, providing an opportunity to correlate endogenous deamidation with function. The large-scale vector preparation protocol requires triple transfection of 293 cells, followed by a 5-day incubation for vector production, and 1-2 days for vector purification. To approximate this process, we prepared a medium-scale triple transfection of 293 cells with wt AAV8 (each in 10x15cm cell culture dishes). Vectors (2x15cm cell culture dishes / day) were collected at 1-day intervals for 5 days, and time points were preserved until the end of the 5-day period by freezing the vectors at -80°C. Crude vector titer and in vitro transduction efficiency were then evaluated as described above. As expected, the number of assembled DNAseI-resistant genomic copies increased over time (Figure 9A). Subsequently, crude vectors were rapidly processed at early (days 1 and 2) and late (day 5) time points by affinity purification, and the in vitro transduction efficiency of huh7 cells was measured. The relative transduction efficiency of the vectors gradually decreased over time (Figure 9B). Regarding transgene expression per GC added to target cells, the vector at day 5 was only 40% of that of the material at day 1. This decrease in activity was also observed in the crude material, indicating a change in the molecular composition before purification (Figure). A similar trend in AAV9 activity loss was observed over 5 days, with the potency of the vector decreasing by approximately 40% (Figures 11I-11K).

[0188] Next, deamidation of time-course samples was measured by mass spectrometry. NG site deamidation progressed substantially over each interval, with an average of 25% deamidation on day 1 and over 60% conversion by day 5 (Figure 9C). Non-NG site deamidation generally progressed over 5 days, but was at much lower levels and less consistent from day 2 to day 5 (Figure 9D). The data correlate endogenous vector deamidation with early time decay at specific activity levels, highlighting the potential opportunity to capture more active vectors by identifying capsid mutations that shorten the production cycle or stabilize asparagine.

[0189] Note that the materials used for mass spectrometry in Figures 2A-2E were at least 7 days post-transfection, as they required an additional 2 days for purification. The higher NG site deamidation (over 80%) in these samples suggests that deamidation likely continues at roughly the same rate after the expression period, as well as 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 low deamidation value is particularly noteworthy when compared to the substance on day 5 (both purified with affinity).

[0190] Stabilizing NG asparagine can improve vector performance. Considering the correlation between vector NG deamidation and transduction efficiency loss, we inferred that stabilizing the NG amide by +1 site mutagenesis might improve vector function. Small-scale vector production was performed for AAV8 NG site mutants in which each +1 residue was individually converted to alanine or serine. Single +1 mutants exhibited good tolerance 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 was also considered. F, et al. (J Virol July 2016;90(16):7196-204) had a flaw in in vitro transduction. The loss of function in the G386 mutant may indicate the selection of deamidated asparagine at N385. Alternatively, the additional side-chain bulk at the +1 position may adversely affect function independently 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, a subset of single-site +1 mutants were tested for hepatic transduction in C57BL / 6 mice. Luciferase expression was investigated by intravenous tail vein infusion (n=3-5) and weekly imaging over two weeks (Figure 10C). In vivo and in vitro transduction data were consistent with the relevant error (i.e., within the margin of error) of each assay. G386 substitution resulted in a loss of transduction, but +1 site mutations at other locations were nearly tolerant and transduced to the liver at levels equal to but not exceeding wtAAV8.

[0191] Since stabilizing the amide at any single NG site may be necessary but insufficient for functional recovery, we then evaluated vector variants with combinations of alanine substitution at the +1 site. We recombined all three AAV8 NG sites (N263, N514, and N540) where the +1 alanine was highly functional. Several combinations, including the triple mutant G264A / G515A / G541A, assembled poorly and were dysfunctional for transduction. However, both paired combinations with N263 (G246A / G515A and G264A / G541A) improved in vitro transduction efficiency (2.0 and 2.6 times, respectively, compared to wtAAV8) and there was no loss of titer (Figure 10D). Since these mutations result in at least two changes (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 models established in time-course studies that NG site deamidation can affect in vitro transduction efficiency.

[0192] Functional asparagine substitution improves lot-to-lot reproducibility in vector manufacturing. Another potentially problematic aspect of the reported vector deamidation profile is the high lot-to-lot variability of deamidation at several sites. For wtAAV8, this variability was most pronounced in N459 (observed deamidation ranged from 0% to 31%) and N499 (observed deamidation ranged from 0% to 53%). Variability in posttranslational modifications is typically virtually avoided during biologic development by completely avoiding clones exhibiting this variability and carefully monitoring and controlling the producing strain and conditions, or by protein engineering of affected candidate proteins.

[0193] Since we were unable to determine the production or processing factors contributing to the 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 substitution to glutamine at each position. Both N459Q and N499Q were efficiently incorporated into the vector, and in vitro transduction was performed. The efficiency was equivalent to that of the wtAAV8 reference (Figure 7A). Next, mutants were produced on a large scale and subjected to mass spectrometry. Consistent with the observation of extremely rare glutamine deamidation, selective and complete stabilization of the glutamine amide at position 459 or 499 was observed in these mutants (data not shown). These mutant lots were evaluated in vivo for hepatic transduction after tail vein injection in C57BL / 6 mice as described above (Figures 7B and 7C). The wTAAV8 vector lot used as a control in this experiment showed 16.8% deamidation at N499, but no deamidation was detected at N459 (data not shown). Hepatic transduction of both mutants at day 14 was equivalent to that of wtAAV8. This data suggests potential protein engineering approaches to address the molecular variability associated with deamidation in the AAV vectors being produced.

[0194] C. Consideration Non-enzymatic deamidation of asparagine and glutamine residues on the AAV8 capsid was independently identified and evaluated using 2D gel electrophoresis, mass spectrometry, novel protein modeling, and functional studies both in vitro and in vivo. Deamidation occurs in a wide variety of proteins and has been shown to significantly affect 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 rotavirus VP6 protein, have been shown to undergo deamidation events by mass spectrometry (Emslie KR et al. Funct Integr Genomics 2000;1(1):12-24).

[0195] The circumstances under which these deamidations occurred in AAV8 suggested that they were the result of spontaneous non-enzymatic events. Asparagine residues are known to be more extensively deamidated than glutamine residues, and the amino acids downstream of asparagine substantially influence the deamidation ratio in which N+1 glycine (i.e., NG) is most efficiently deamidated. We observed a striking confirmation of the role of N+1 amino acids in the deamidation of AAV capsids, with all NGs present in VP1 being deamidated at levels exceeding 75%, while deamidation consistently did not exceed 20% for any other asparagine or glutamine in the capsid. Substantially all NG motifs in AAV8 and AAV9 capsids (i.e., 7 / 9) were also present on the surface of the capsid, which is located in the HVR region associated with higher-order structural flexibility and thermal vibrations. This is consistent with previous reports of NG motifs in other proteins located in regions where flexibility may be required for proper protein function, and not in 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 surface-exposed NG motifs in HVRs further improves the deamidation rate by providing solvent exposure and conformational flexibility, thereby facilitating the formation of succinimidyl intermediates. As predicted, unfavorable environments lead to a much lower deamidation rate.

[0196] A key question regarding the biology of AAV and its use as a vector is the functional consequences of these deamidations. Mutations in the capsid DNA that convert asparagine to aspartate allow for the evaluation of capsids where all amino acids at a particular site are represented as aspartate. However, there are no easy strategies to prevent deamidation using mutagenesis other than potentially muting the N+1 residue, which can be confused with the direct consequences of a second site mutation. We studied a limited number of variants in which the asparagine residue is converted to aspartate by mutagenesis. Functional analysis includes capsid assembly as well as in vitro and in vivo transduction. The most substantial impact of mutagenesis on vector function is observed in those containing asparagine that is incompletely deamidated at baseline and whose surface is not exposed. However, surprisingly, mutagenesis of highly deamidated asparagine at 514 to aspartic acid had some effect on function. This result suggests that the presence of a residual amount of the corresponding amide may affect function. This may be partly due to the presence of hydrogen bonding interactions between N514 and D531 of another triple-related VP3 monomer (identified in the wtAAV8 crystal structure) that is lost when this residue is converted to aspartic acid after deamidation.

[0197] When evaluating the impact of these deamidations on the development of novel therapeutics, it is important to better understand the factors influencing the degree of deamidation in AAV vectors. Incubation of the vector under extreme conditions known to significantly accelerate deamidation kinetics had little effect. Combined with isotopic incorporation studies, these results suggest that deamidation occurs during capsid assembly and is not an artifact of vector processing or mass spectrometry. Deamidation at the NG site is unlikely to have a substantial impact on vector performance, as the reaction was virtually complete in all samples evaluated. However, initial functional studies suggest that residual amounts of undeamidated asparagine may contribute to function. While there are concerns about sites with low deamidation completeness, these were mostly related to inter-sample variability. One example is asparagine at position 499, which showed deamidation ranging from 0% to 53% with an average of 17%. Subtle differences in vector production conditions may contribute to this heterogeneity. The striking similarity in deamidation between AAV8 and AAV9 suggests that this is a characteristic of the entire virus family.

[0198] In summary, we discovered substantial heterogeneity in the primary amino acid structures of AAV8 and AAV9 capsid proteins. These studies have potential implications for the development of AAV as a vector in several ways. Firstly, the actual amino acid sequence of the VP protein is not predicted by the corresponding DNA sequence. Secondly, the manner of production can lead to deamidation variations and corresponding changes in vector function. Until we understand the factors influencing the deamidation ratio at non-NG sites and better understand its functional consequences, deamidation may need to be included in the characterization of clinical-grade AAV vectors. While 2D gel electrophoresis can provide an overall assessment of net deamidation, mass spectrometry would likely be necessary to evaluate deamidation at specific residues.

[0199] Example 2: Deamidation of AAV5.5.9 Novel sequences of AAV5.5.9 are provided in SEQ ID NOs. 9 and 10, respectively. The AAV5.5.9 vector was evaluated for deamidation as described in Example 1 for AAV9. Highly deamidated residues were found at N57, N319, N442, and N502. [Table 10]

[0200] Example 3: Deamidated AAVrh79 (clade E) AAVrh79 was isolated from DNA extracted from the small intestinal tissue of rhesus monkeys. It is characterized as being systematically within clade E (Figures 14A-14D). Its sequence is provided herein, with the nucleotide sequence in SEQ ID NO: 1 and the amino acid sequence in SEQ ID NO: 2. The amino acid sequence alignments of AAVrh79, AAVrh.10, and AAVhu.37 are provided in Figure 14A. The nucleic acid sequence alignments of AAVrh79, AAVrh.10, and AAVhu.37 are provided in Figures 14B-14D.

[0201] AAVrh79 has three amino acid differences in its primary sequence. AAVhu37 has Ala at position 67 and Lys at position 169 in its primary VP1 sequence, while AAVrh79 has glutamic acid (E) at position 67 and Arg at position 169. The differences in VP1 DNA sequences between Rh.79, hu.37, and hu.40 are shown in Figure 11B. Vectors expressing eGFP were prepared based on various clade E variants and their relative infectivity to Huh7 cells was evaluated (Figure 11C). C57BL / 6 mice were given two dose levels (3 x 10) of either eGFP-expressing AAV8 or AAVrh.79 vectors. 10 and 3x10 11 GC / mouse cells were injected, and infectivity was evaluated by fluorescence microscopy (data not shown).

[0202] Vectors based on AAVrh79 were prepared for the production of caps using the AAVrh79 nucleotide sequence using known production techniques, for example, as described above for AAV8 vectors. The results of production yield and production purity evaluations are shown in FIGS. 15A to FIGS. 15B and 16, respectively.

[0203] To evaluate the expression level using AAVrh79 containing a marker gene (firefly luciferase), male RAG KO mice at 6-8 weeks of age were injected intramuscularly with 3×10 11 GC / mouse vectors using a Hamilton syringe. Expression of ffLuc was visualized by whole-body bioluminescence imaging as described above [Greig JA, Peng H, Ohlstein J, Medina-Jaszek CA, Ahonkhai O, Mentzinger A, et al. (2014) Intramuscular Injection of AAV8 in Mice and Macaques Is Associated with Substantial Hepatic Targeting and Transgene Expression. PLoS ONE 9(11):e112268. doi.org / -10.1371 / journal.pone.-0112268.]. The results are shown in FIGS. 17A-17D.

[0204] Expression of AVV8 triple, AAVhu68, AAV9, AAV8, and AAVrh79 vectors was compared after intramuscular administration of 10 13 GC / kg AAVrh79 to male and female cynomolgus macaques (FIG. 17E).

[0205] Vectors expressing the secreted transgene (201Ig IA) were administered to male RAG KO mice (n = 5 / group) (3×10 10 or 3×10 11The vector was administered intramuscularly into the gastrocnemius muscle of GC / mice. The results showed that AAV8 triple was better expressed after IM injection, and at the lower doses tested, the difference in expression from AAV8 triple was substantial. At higher doses, AVVrh79 was expressed at levels comparable to the other vectors tested (Figure 17F).

[0206] Female cynomolgus macaques (RA2362) were pre-screened for NAb (Figure 18A), and AAVG2, TBG, eGFP, WPRE, bGH (1x10) were selected. 13 ddGC / kg (intravenous) was injected. Seven days after treatment, the animals were euthanized, and necropsies were performed to isolate the liver and other tissues for analysis. GFP expression in the liver and spleen was evaluated on day 7 (Figures 18B and 18C). Figure 18D shows the levels of vectors detected in various tissues. GFP expression levels were evaluated in liver animals treated with AAVG2 (RA2362) or AAV8 and AAVG3 vectors (Figures 18E to IJ). Figure 18K shows the levels of vectors detected in various tissues from these animals.

[0207] Various AAV8 and AAVrh79 vectors were generated, and in some cases, multiple lots were produced. The yields of these AAV9 and AAVrh79 vector lots were compared (Figure 19).

[0208] The AAVrh79 vector was evaluated for deamidation for AAV8 and AAV9 as described in Example 1. The results indicate that the vector contains four highly deamidated amino acids (N57, N263, N385, N514) corresponding to the asparagine in the asparagine-glycine pair, based on the numbering of AAVrh79 (SEQ ID NO: 1). Lower deamidation rates were consistently observed at residues N94, N254, and N410. [Table 11-1] [Table 11-2]

[0209] Example 4: Preparation of AAV8.2.08 As discussed in WO 2017 / 180854 (incorporated herein by reference), several AAV8 variants were generated with mutations in the HVR.VIII region at c41, c42, c46, g110, g113, g115, and g117. As discussed by Gurda et al., the major ADK8 epitope is located in the HVR.VIII region (amino acids 586-591 using AAV8 vp1 numbering). These variants were tested in vitro for ADK8 resistance, and some of them were tested in vivo for ADK8 resistance. See, for example, Lochrie 2006 above.

[0210] AR2.1-9 were randomly selected. AR2.25-61 were selected based on frequency. The randomly selected variants, as well as their high-frequency variants, survived in terms of 6-well plate yield and Huh7 transduction. Figure 24 shows the expression of AAV8.AR2.08 in various tissues (most sets on the left of the bars).

[0211] Example 5: Deamidation of AAV8.AR2.08 Novel sequences of AAV8.AR2.08 were provided as sequence numbers 17 and 18, respectively, and these were designed in the same manner as in Example 4.

[0212] A. A modified AAV8.AR2.08 vector was prepared, and the modifications to AAV8 were evaluated as described in Example 1. The results show that the vector contains five highly deamidated amino acids (N57, N263, N385, N514, and N540) corresponding to the asparagine in the asparagine-glycine pair, based on the numbering of AAV8.AR2.08 (SEQ ID NO: 18). Lower deamidation rates were consistently observed at residues N94, N254, and N410. In contrast to AAV8, deamidation was not observed at position N459 (average 7% in AAV8) or N499 (average 17% in AAV8). [Table 12-1] [Table 12-2]

[0213] B. Single-cell RNA-seq reveals the tissue localization and transcriptional signatures of transduced hepatocytes isolated from non-human primates after treatment with AAV8. Single-cell RNA sequencing has proven to be a powerful technique for characterizing cellular transcriptomes with unprecedented single-cell resolution. In this study, we utilize single-cell RNA-seq to investigate the transcriptional landscape of primary hepatocytes isolated from rhesus monkeys after treatment with a GFP-expressing AAV8 vector. Transcriptome analysis of GFP+ and GFP- cells sorted by FACS will reveal the tissue localization of transdextrins within hepatic lobules, as well as the genes and regulatory pathways involved in hepatocyte transduction and transgene expression.

[0214] For the study design, rhesus macaques were treated with either 1x10¹³ ddGC / kg AAV8.TBG.EGFP.WPRE (n=1) or 1x10¹³ ddGC / kg AAV8.2.08.TBG.EGFP.WPRE (AAV8 variant, n=1). Seven days after treatment, the animals were euthanized, and necropsy was performed to isolate the livers from both animals. After treatment with collagenase and gradient centrifugation, the isolated liver cells were FACS sorted on BD Precise® 96-well plates by GFP transgene expression. 192 single cells were isolated from each animal (96GFP+ and 96GFP-) and subsequently used to prepare single-cell RNA-seq libraries according to the standard BD Precise® protocol. We determined differentially expressed transcripts between GFP- and GFP+ sorted cells and analyzed the data using the Seurat, Scran, and Scater packages in R to perform spatial reconstruction of isolated cells within liver lobules using the established transcriptional expression signatures.

[0215] We found that AAV8.AR2.08 exhibited increased liver-specification and a 1.5-fold increase in transduction efficiency compared to AAV8. Single-cell transcriptome analysis of selected liver cells revealed that transgene-expressing cells were evenly distributed throughout the hepatic lobule, with a slight preference for the periportal region, as observed by histopathology. Interestingly, we found that a subpopulation of selected GFP- cells expressed transgene transcripts at levels comparable to those of selected GFP+ cells, suggesting that these cells were indeed transduced and expressed transgene mRNA despite the absence of detectable levels of translational protein. Comparing the transcriptional profiles of GFP- and GFP+ cells revealed different expressed transcripts involved in viral mRNA translation and elucidated pathways potentially involved in regulating transgene protein expression in transduced cells.

[0216] C. Isolation of adeno-associated virus 8 variants with better liver transduction and higher liver specificity in non-human primates with directed evolution via a human liver xenograft model. To derive AAV variants with better transduction and higher specificity, AAV-directed evolution was performed using saturated mutagenesis targeting the surface-exposed site on the AAV8 capsid, a benchmark for liver gene therapy, followed by two rounds of in vivo enrichment in a human liver xenograft mouse model, isolating an AAV8 variant designated AAV8.2.08. After intravenous injection into non-human primates at a dose of 1e13 genome copies (GC) / kg body weight, delivery in the liver increased compared to AAV8, while the vector genome copies delivered by AAV8.AR2.08 decreased in various organs (including lungs, heart, stomach, pancreas, kidneys, and mesenteric lymph nodes), indicating better liver transduction and higher tissue specificity. Next-generation sequencing showed significant enrichment of AAV8.AR2.08 during in vivo selection, suggesting a potential approach for isolating capsids with novel and improved directivity (other comparisons are shown in Figures 21–27). Figure 28 shows the biological distribution of AAV8.AR2.08 and AAV8.

[0217] D. Barcoding Six black mice were given a mixture of rAAVG3 derived from 12 preparations, 2 × 10⁶ times. 12GC / mice were intravenously injected (Figure 18B). Each preparation contained a distinct barcode within the vector genome, allowing for identification of a specific preparation (Figure 18A). After two weeks, the animals were euthanized and tissues were collected. As predicted, rAAVG3 expression in the liver was higher than in the heart or muscle. Figure 18C shows that the tissue distribution experiment indicates that the actual frequencies are consistent with the theoretical frequencies of barcodes in the injected vector mixture (Figure 18D, total; Figures 19A, 19B, muscle; Figures 19C, 19D, heart; and Figures 19E, 19F, liver), with slight abnormalities in BC02 and BC06 (Figures 20A-20C). All documents cited herein are incorporated herein by reference. U.S. Provisional Patent Applications No. 62 / 722,388 and No. 62 / 722,382, both filed on 24 August 2018; U.S. Provisional Patent Applications No. 62 / 703,670 and No. 62 / 703,673, both filed on 26 July 2018; U.S. Provisional Patent Applications No. 62 / 677,471 and No. 62 / 677,474, both filed on 29 May 2018; U.S. Provisional Patent Application No. 62 / 667,585, filed on 29 May 2018; and U.S. Provisional Patent Application No. 62 / 635,964, filed on 27 February 2018 are incorporated herein by reference. The following U.S. Provisional Patent Applications, No. 62 / 667,881, No. 62 / 667,888, No. 62 / 667,888, No. 62 / 667,587, No. 62 / 663,797, No. 62 / 663,788, and No. 62 / 635,968, filed on May 6, 2018, No. 62 / 663,797, No. 62 / 663,788, and No. 62 / 635,968, filed on April 27, 2018, are incorporated by reference. The sequence numbers referenced herein and shown in the accompanying sequence listings are incorporated by reference. While the present invention is 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 accompanying claims.

Claims

1. The use of recombinant AAVrh79 (rAAVrh79) for the manufacture of pharmaceuticals useful for the delivery of gene products to target cells, wherein the rAAVrh79 is (A) AAVrh79 vp1 protein, AAVrh79 vp2 protein, and AAVrh79 vp3 protein, which are products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, wherein the AAVrh79 vp1 protein contains glutamic acid (E) at position 67 and arginine (R) at position 169 according to SEQ ID NO: 2, and the AAVrh79 vp2 protein contains arginine (R) at position 169 of vp2 according to SEQ ID NO: 2, (B) A vector genome in the AAVrh79 capsid, wherein the vector genome comprises a nucleic acid molecule comprising a non-AAV nucleic acid sequence encoding the product, which is operably linked to an AAV reverse terminal repeat sequence and a sequence that directs the expression of the product in target cells, The aforementioned use, including.

2. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to claim 1, wherein the nucleic acid sequence encoding the AAVrh79 capsid protein is Sequence ID No.

1.

3. The use of recombinant AAVrh79 (rAAVrh79) for the manufacture of pharmaceuticals useful for the delivery of gene products to target cells, wherein the rAAVrh79 is (A) A heterogeneous population of AAVrh79 vp1 protein, AAVrh79 vp2 protein, and AAVrh79 vp3 protein, each containing amino acids 1-738 (vp1), 138-738 (vp2), and 204-738 (vp3) of SEQ ID NO: 2, wherein the AAVrh79 vp1 protein contains glutamic acid (E) at position 67 of vp1 and arginine (R) at position 169 according to SEQ ID NO: 2, and the AAVrh79 vp2 protein contains arginine (R) at position 169 of vp2 according to SEQ ID NO: 2, and the AAVrh79 vp1 protein, the AAVrh79 vp2 protein, and the AAVrh79 The vp3 protein contains asparagine (N) that is 50% to 100% deamidated at at least two of the N57, N263, N385, and N514 positions of SEQ ID NO: 2, as determined by mass spectrometry, and as a result of the deamidation, an amino acid change occurs to aspartic acid, isoaspartic acid, interconverted aspartic acid / isoaspartic acid pairs, or combinations thereof, and the heterogeneous population, (B) A vector genome in the AAVrh79 capsid, wherein the vector genome comprises a nucleic acid molecule comprising a non-AAV nucleic acid sequence encoding the product, which is operably linked to an AAV reverse terminal repeat sequence and a sequence that directs the expression of the product in target cells, The aforementioned use, including.

4. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for the delivery of a gene product to a target cell, according to claim 3, wherein the AAVrh79 vp1 protein, the AAVrh79 vp2 protein, and the AAVrh79 vp3 protein further comprise one or more of acetylated lysine, phosphorylated serine and / or threonine, isomerized aspartic acid, deamidated glutamine, oxidized tryptophan and / or methionine, or amidated amino acids.

5. The AAVrh79 capsid is (a) When determined by mass spectrometry, based on the numbering in Sequence ID No. 2, at least 75% of the asparagine (N) in the asparagine-glycine pair located at position N57 of the vp1 protein is deamidated. (b) When determined by mass spectrometry, based on the residue numbering of the amino acid sequence of Sequence ID No. 2, at least 75% of the nitrogen atoms in the asparagine-glycine pair at position N263 of the vp1 protein, the vp2 protein, and the vp3 protein are deamidated. (c) As determined by mass spectrometry, based on the residue numbering of the amino acid sequence of Sequence ID No. 2, at least 70% of the nitrogen in the asparagine-glycine pair at position N385 of the vp1 protein, the vp2 protein, and the vp3 protein is deamidated, and / or (d) When determined by mass spectrometry, based on the residue numbering of the amino acid sequence of Sequence ID No. 2, at least 85% of the nitrogen atoms in the asparagine-glycine pair at position N514 of the vp1 protein, the vp2 protein, and the vp3 protein are deamidated. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to claim 3 or 4, comprising one or more of the above.

6. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 3 to 5, wherein the rAAVrh79 capsid comprises the vp1 protein in which 75% to 100% of the N at position N57 of the AAVrh79 vp1 protein is deamidated as determined by mass spectrometry.

7. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 3 to 6, wherein the rAAVrh79 capsid comprises AAVrh79 vp1 protein, AAVrh79 vp2 protein, and / or AAVrh79 vp3 protein, in which 75% to 100% of the N at position N263 and / or N385 is deamidated based on the numbering of Sequence ID No. 2 when determined by mass spectrometry.

8. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 3 to 7, wherein the rAAVrh79 capsid comprises vp1 protein, vp2 protein, and / or vp3 protein, in which 75% to 100% of the N at position N514 is deamidated based on the numbering of Sequence ID No. 2 as determined by mass spectrometry.

9. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 3 to 8, wherein the rAAVrh79 capsid further comprises at least subpopulations of vp1 protein, vp2 protein, and / or vp3 protein having amino acid modifications from SEQ ID NO: 2, including 50-100% deamidation at positions N57, N263, N385, and N514, respectively, as determined by mass spectrometry.

10. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 3 to 9, wherein the rAAVrh79 capsid comprises a subpopulation of vp1 protein, vp2 protein, and / or vp3 protein, further comprising 1% to 40% deamidation at one or more of the N94, N254, N305, N410, N479, N653 positions, or combinations thereof, as determined by mass spectrometry.

11. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 3 to 10, wherein the AAVrh79 vp1 protein, the AAVrh79 vp2 protein, and the AAVrh79 vp3 protein have less than 10% deamidation at positions N94, N254, N305, N410, N479, and N653, less than 5% methylation, and / or less than 5% isomerization.

12. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to target cells, according to any one of claims 3 to 11, further comprising 1% or less of deamidated glutamine(optional) which is deamidated to (α)-glutamic acid, γ-glutamic acid, interconverted (α)-glutamic acid / γ-glutamic acid pairs, or combinations thereof.

13. Use of rAAVrh79 for the manufacture of a pharmaceutical useful for delivery of a gene product to a target cell, according to any one of claims 1 to 12, wherein the AAV ITR sequence is a 5' ITR and a 3' ITR derived from AAV2.

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