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

Abstract

The present application provides novel adeno-associated virus (AAV) vectors, AAV vectors with reduced capsid deamidation, and uses thereof. A recombinant adeno-associated virus (rAAV) vector includes an AAV capsid having a heterogeneous population of vp1 protein, a heterogeneous population of vp2 protein, and a heterogeneous population of vp3 protein. In particular, the present invention relates to a recombinant VP1 amino acid sequence comprising a capsid comprising a modified amino acid as compared to the encoded VP1 amino acid sequence, the capsid comprising a highly deamidated asparagine residue at an asparagine-glycine pair, and further comprising a plurality of other less deamidated asparagine residues, and optionally a glutamine residue.

Description

[0001] This application is a divisional application of the invention application with the application date of February 27, 2019, the Chinese national application number being 201980029022.1, and the invention name being “Novel adeno-associated virus (AAV) vector, AAV vector with reduced capsid deamidation and its use”. Technical Field

[0002] The present invention relates to the field of adeno-associated virus (AAV) vectors. In particular, the present invention provides a recombinant AAV vector having a capsid that maintains greater stability, potency and / or purity. Background Art

[0003] The structure of the adeno-associated virus (AAV) capsid is icosahedral and contains 60 viral protein (VP) monomers (VP1, VP2 and VP3) in a ratio of 1:1:10 (Xie Q et al., Proc Natl Acad Sci USA 2002; 99(16):10405-10). Both VP1 and VP2 contain the entire VP3 protein sequence (approximately 535 aa) within their C-termini, and the shared VP3 sequence is primarily responsible for the overall capsid structure. Due to the structural flexibility of the VP1 / VP2 unique region and the low representation of VP1 and VP2 monomers relative to VP3 monomers in the assembled capsid, VP3 is the only capsid protein that can be resolved by x-ray crystallography (Nam HJ et al., J Virol 2007; 81(22):12260-71). VP3 contains nine hypervariable regions (HVRs) that are the main source of sequence variation between AAV serotypes (Govindasamy L et al., J. Virol. 2013; 87(20): 11187-99). Given their flexibility and location on the capsid surface, HVRs are primarily responsible for interacting with target cells and the immune system (Huang LY et al., J. Virol. 2016; 90(11): 5219-30; Raupp C et al., J. Virol. 2012; 86(17): 9396-408). Although the structures of various serotypes are published (Protein Data Bank (PDB) IDs 1LP3, 4RSO, 4V86, 3UX1, 3KIC, 2QA0, 2G8G from the Research Collaboration for Structural Bioinformatics (RCSB) database for structural entries for AAV2, AAVrh.8, AAV6, AAV9, AAV3B, AAV8, and AAV4, respectively), there is little information in the literature regarding modifications on the surface of these capsids. Studies have shown that intracellular phosphorylation of the capsid occurs at specific tyrosine residues (Zhong L et al., Virology 2008; 381(2): 194-202). Despite the presence of putative glycosylation sites in the primary VP3 sequence, glycosylation events have not been identified in AAV2 (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 been evaluated for capsid glycosylation.

[0004] AAV gene therapy vectors have undergone less molecular scrutiny than is typically associated with the development and manufacture of recombinant protein therapeutics. AAV capsid post-translational modifications (PTMs) remain largely unexplored, and thus, little is known about their potential to impact function or about strategies to control PTM levels in manufactured AAV therapeutics.

[0005] Variation in post-translational modifications of non-gene therapy protein therapeutics complicates their development as drugs. Jenkins, N, Murphy, L, and Tyther, R (2008), “Post-translational modifications of recombinant proteins: significance for 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 anthrax vaccines based on recombinant protective antigens. (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 to modulate host cell signaling pathways or innate immune responses (Zhao J et al., Journal of Virology 2016; 90(9):4262-8; Zhao J et al., Cell Host Microbe 2016; 20(6):770-84). More commonly, endogenous deamidation is an enzyme-independent, spontaneous process. Although the purpose of spontaneous deamidation has not been fully elucidated, previous studies have suggested that this event serves as a molecular clock to indicate the relative age of proteins and regulate their 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 commonly, glutamine undergoes nucleophilic attack from an adjacent nitrogen atom and the amide group is lost. This process produces a succinimidyl intermediate (Yang H and Zubarev RA, Electrophoresis 2010; 31(11): 1764-72), which decomposes by hydrolysis to a mixture of aspartic acid and isoaspartic acid (or glutamic acid and isoglutamic acid) (Catak S et al., J Phys Chem A 2009; 113(6): 1111-20). Studies of short synthetic peptides estimate that this hydrolysis produces a mixture of isoaspartic acid and aspartic acid in a ratio of 3:1 (Geiger T. and Clarke S, J Biol Chem 1987; 262(2): 785-94).

[0007] There remains a need for compositions comprising AAV-based constructs to deliver heterologous molecules with stable receptor binding and / or stable capsids to avoid neutralizing antibodies and / or maintain purity upon storage. Summary of the Invention

[0008] In one embodiment, a composition is provided, comprising a mixed population of recombinant adeno-associated viruses (rAAVs), each of the rAAVs comprising: (a) an AAV capsid, the AAV capsid comprising about 60 capsid vp1 proteins, vp2 proteins, and vp3 proteins, wherein the vp1 proteins, the vp2 proteins, and the vp3 proteins are a heterogeneous population of vp1 proteins, the vp1 proteins being selected from the AAVs. a heterogeneous population of vp2 proteins produced by a nucleic acid sequence encoding a selected AAV vp2 amino acid sequence; a heterogeneous population of vp3 proteins produced by a nucleic acid sequence encoding a selected AAV vp3 amino acid sequence, wherein the vp1 proteins, the vp2 proteins, and the vp3 proteins contain a subpopulation having amino acid modifications comprising at least two highly deamidated asparagines (N) of asparagine-glycine pairs in the AAV capsid, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (b) a vector genome in the AAV capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product, the non-AAV nucleic acid sequence being operably linked to a sequence that directs expression of the product in a host cell.

[0009] In certain embodiments, the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof. In certain embodiments, the capsid further comprises one or more deamidated glutamines, and the one or more deamidated glutamines are deamidated to (α)-glutamic acid, γ-glutamic acid, an interconverting (α)-glutamic acid / γ-glutamic acid pair, or a combination thereof.

[0010] In another aspect, a recombinant adeno-associated virus (rAAV) is provided, the rAAV comprising: (A) an AAVrh79 capsid comprising one or more of the following: (1) AAVrh79 capsid proteins comprising: a heterogeneous population of AAVrh79 vp1 proteins selected from the group consisting of: vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2, vp1 proteins produced from SEQ ID NO: 1, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2 that is at least 70% identical to SEQ ID NO: 1; and a heterogeneous population of AAVrh79 vp2 proteins selected from the group consisting of: vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2, vp1 proteins produced from SEQ ID NO: 1, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2 that is at least 70% identical to SEQ ID NO: 1. 1 to 2214 of SEQ ID NO: 1; or a heterogeneous population of AAVrh79 vp3 proteins selected from the group consisting of: vp3 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 2; vp3 proteins produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 1; or vp2 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 2 that is at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO: 1. 2; and / or (2) a heterogeneous population of vp1 proteins produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO:2; and / or (3) a heterogeneous population of vp2 proteins produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO:2;and a heterogeneous population of vp3 proteins, the vp3 proteins being products of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO:2, wherein: the vp1 proteins, the vp2 proteins, and the vp3 proteins contain a subpopulation having amino acid modifications comprising at least two highly deamidated asparagines (N) of the asparagine-glycine pairs in SEQ ID NO:2, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector genome in the AAVrh79 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product, the non-AAV nucleic acid sequence being operably linked to a sequence that directs expression of the product in a host cell.

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

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

[0013] In another embodiment, a rAAV8.AR2.08 is provided, comprising: (A) an AAV8.AR2.08 capsid, the AAV8.AR2.08 capsid comprising one or more of the following: (1) AAV8.2.08 capsid proteins, the AAV8.2.08 capsid proteins comprising: a heterogeneous population of AAV8.AR2.08 vp1 proteins, the AAV8.AR2.08 vp1 proteins being selected from the group consisting of: vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 18, vp1 proteins produced from SEQ ID NO: 17, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 18 that is at least 70% identical to SEQ ID NO: 17; and a heterogeneous population of AAV8.AR2.08 vp2 proteins being selected from the group consisting of: vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 18, vp1 proteins produced from SEQ ID NO: 17, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 18 that is at least 70% identical to SEQ ID NO: 17. AAV8.AR2.08 vp2 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 18, a vp2 protein produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 17, or a vp2 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 18 that is at least 70% identical to at least nucleotides 411 to 2214 of SEQ ID NO: 17; and a heterogeneous population of AAV8.AR2.08 vp3 proteins, the AAV8.AR2.08 vp3 proteins being produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 17.08 vp3 protein is selected from the group consisting of: vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 18, vp3 protein produced from a sequence comprising at least nucleotides 607 to 2214 of SEQ ID NO: 17, or vp3 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 18 that is at least 70% identical to at least nucleotides 607 to 2214 of SEQ ID NO: 17; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 18; and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18. NO:18, wherein: the vp1 protein, the vp2 protein, and the vp3 protein contain a subpopulation with amino acid modifications, the amino acid modifications including at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:18, and optionally further include a subpopulation containing other deamidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector genome in the AAV8.AR2.08 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding the product, the non-AAV nucleic acid sequence being operably linked to a sequence that directs expression of the product in a host cell.

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

[0015] In yet another aspect, a method for reducing deamidation of an AAV8.AR2.08 capsid is provided. In one embodiment, the method comprises generating an AAV8.AR2.08 capsid from a nucleic acid sequence comprising a modified AAV8.AR2.08 vp codon, the nucleic acid sequence comprising independently modified glycine codons at one to four of the asparagine-glycine pairs located at positions N57, N263, N385, N514, and / or N540 of SEQ ID NO: 18, such that the modified codons encode an amino acid other than glycine. In another embodiment, the method comprises producing an AAV8.AR2.08 capsid from a nucleic acid sequence comprising modified AAV8.AR2.08 vp codons, comprising independently modified glycine codons at 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 certain embodiments, a rAAV5.5.9 is provided, comprising: (A) an AAV5.5.9 capsid, the AAV5.5.9 capsid comprising one or more of the following: (1) an AAVG5 capsid protein, the AAVG5 capsid protein comprising: a heterogeneous population of AAV5.5.9 vp1 proteins, the AAV5.5.9 vp1 proteins being selected from the group consisting of: vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 10, vp1 proteins produced from SEQ ID NO: 9, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 1 that is at least 70% identical to SEQ ID NO: 9; and a heterogeneous population of AAV5.5.9 vp2 proteins, the AAV5.5.9 vp2 proteins being selected from the group consisting of: vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 10, vp1 proteins produced from SEQ ID NO: 9, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 1 that is at least 70% identical to SEQ ID NO: 9. AAV5.5.9 vp3 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:10, a vp2 protein produced from a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO:9, or a vp2 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:10 that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:9; and an AAV5.5.9 vp3 protein selected from the group consisting of: an AAV5.5.9 vp3 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:10.9vp3 proteins, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2211 of SEQ ID NO:9, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO:9 and encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:10; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:10; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:10; and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 203 to 726 of SEQ ID NO:10, wherein: the vp1 proteins, the vp2 proteins, and the vp3 proteins contain subpopulations having amino acid modifications comprising SEQ ID NO:10. at least two highly deamidated asparagines (N) in the asparagine-glycine pair of ID NO: 10, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector genome in the AAV5.5.9 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product, the non-AAV nucleic acid sequence being operably linked to a sequence that directs expression of the product in a host cell.

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

[0018] In yet another aspect, a method for reducing deamidation of an AAV5.5.9 capsid is provided. In one embodiment, the method comprises generating an AAV5.5.9 capsid from a nucleic acid sequence comprising a modified AAV5.5.9 vp codon, the nucleic acid sequence comprising independently modified glycine codons at one to four of the asparagine-glycine pairs located at positions N57, N319, N442, and / or N502 of SEQ ID NO: 10, such that the modified codons encode an amino acid other than glycine. In another embodiment, the method comprises producing an AAV5.5.9 capsid from a nucleic acid sequence comprising modified AAV5.5.9 vp codons, wherein the nucleic acid sequence includes independently modified glycine codons at one to four 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 aspect, 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 aspect, provided is a recombinant AAV (rAAV) as described herein for use in delivering a desired gene product to a subject in need thereof.

[0021] In another aspect, an rAAV production system is provided for producing rAAV as described herein. In one embodiment, the system comprises: (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 the AAV capsid, the nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product, the non-AAV nucleic acid sequence being operably linked to a sequence that directs expression of the product in a host cell; and (c) AAV rep functions and helper functions sufficient to allow packaging of the nucleic acid molecule into a recombinant AAV capsid.

[0022] These and other aspects of the invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A-1G : Electrophoretic analysis of AAV8 VP isoforms. ( Figure 1A The diagram shows the mechanism by which an asparagine residue undergoes nucleophilic attack by the adjacent nitrogen atom to form a succinimidyl intermediate. This intermediate then undergoes hydrolysis, decomposing into a mixture of aspartic acid and isoaspartic acid. The β carbon is labeled as such. The diagram was generated in BIOVIADraw 2018. Figure 1B ) 1 μg of AAV8 vector was run on denaturing one-dimensional SDS-PAGE. ( Figure 1C ) shows the isoelectric point of the carbonic anhydrase pI marker point. ( Figure 1D 5 μg of AAV8 vector was analyzed by two-dimensional gel electrophoresis and stained with Coomassie blue. Spots 1-20 are carbamylated carbonic anhydrase pI markers. Boxed regions are as follows: a = VP1, b = VP2, c = VP3, d = internal tropomyosin marker (arrow: tropomyosin spot with MW = 33 kDa, pI = 5.2). Isoelectric focusing was performed over a pI range of 4-8. Figure 1E-1G ) Results of isoelectric focusing performed in the pI range of 4-8. 1e11 GC of wtAAV8 vector ( Figure 1E ) or mutation vector ( Figure 1F and Figure 1G ) and stained with Sypro Ruby. Protein markers: A = VP1; B = VP2; C = VP3, D = hen egg white conalbumin marker, E = turbouclease marker. Isoelectric focusing was performed over a pI range of 6-10. Primary VP1 / 2 / 3 isoform spots are circled, and the migration distance of the primary marker spot is indicated by a vertical line (turbonuclease = dashed line, conalbumin = solid line).

[0024] Figure 2A-2E : Analysis of asparagine and glutamine deamidation in AAV8 capsid protein. Figure 2A-2B ) shows the electrospray ionization (ESI) mass spectrum and the Asn-94 ( Figure 2A ) and Asp-94( Figure 2B Theoretical and observed masses of the 3+ peptide (93-103). Figure 2C-2D ) shows the ESI mass spectrum and the presence of Asn-254 ( Figure 2C ) and Asp-254( Figure 2D Theoretical and observed masses of the 3+ peptide (247-259) of ). The observed mass shifts of Asn-94 and Asn-254 are 0.982 Da and 0.986 Da, respectively, while the theoretical mass shift is 0.984 Da. ( Figure 2E) Shows the percentage of deamidation at specific asparagine and glutamine residues of interest for AAV8 tryptic peptides purified by different methods. Bars indicating deamidation at asparagine residues with N+1 glycine are cross-hatched. Residues determined to be at least 2% deamidated in at least one of the preparations analyzed are included. Data are expressed as mean ± standard deviation.

[0025] Figure 3A-Figure 3E :Structural modeling of AAV8 VP3 monomer and analysis of deamidation sites. ( Figure 3A ) shows the AAV8 VP3 monomer (PDB identifier: 3RA8) in a coiled representation. The color of the ribbon indicates the relative degree of flexibility (blue = most rigid / normal temperature coefficient, red = most flexible / high temperature coefficient). Spheres indicate residues of interest. The expanded view is a ball and stick representation of the residue of interest and its surrounding residues to show the local protein structure (blue = nitrogen, red = oxygen). Underlined residues are residues in the NG motif. Figure 3B-3E : shows the isoaspartate model of asparagine with N+1 deamidation of glycine. Figure 3C )N263、( Figure 3D )N514 and ( Figure 3E )N540 isoaspartate model compared with ( Figure 3B ) The asparagine model of N410 generated a 2FoFc electron density map (1σ level) from refinement of the AAV8 crystal structure (PDB ID: 3RA8). The electron density map is shown as a magenta grid. The β carbon is labeled as such. Arrows indicate electron density corresponding to the R group of the residue of interest.

[0026] Figures 4A-4D : Determination of factors affecting AAV8 capsid deamidation. Figure 4A ) were incubated at 70°C for three or seven days, ( Figure 4B ) were exposed to pH 2 or pH 10 for seven days, or prepared using D2O instead of H2O ( Figure 4C ) for mass spectrometry to identify possible sources of deamidation that are not intrinsic to AAV capsid formation. ( Figure 4D ) using B1 antibody (reacting with denatured capsid) and AAV8 conformation-specific antibody (reacting with intact capsid) as Figure 4A Dot blots of the vectors were processed as such to assess capsid structural integrity.

[0027] Figure 5A-5B : Deamidation frequency in non-AAV proteins. For comparison with AAV deamidation percentages, two non-AAV recombinant proteins containing the NG motif that may be deamidated (human carbonic anhydrase ( Figure 5A) and rat phenylalanine-hydroxylase ( Figure 5B )) deamidation percentage.

[0028] Figure 6 : Comparison of AAV8 deamidation percentages calculated using data analysis pipelines from two institutions. Shown are the deamidation percentages of AAV8 tryptic peptides assessed at two different institutions at specific asparagine and glutamine residues of interest.

[0029] Figures 7A-7C Demonstrates functional asparagine substitutions at non-NG sites with high variability between batches. Figure 7A ) The titers of wtAAV8 vector and mutant vectors were generated by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). The titers are reported relative to the wtAAV8 control. Figure 8B Transduction efficiency was measured as described in . Titers and transduction efficiencies were normalized to the values ​​of the wtAAV8 control. ( Figure 7B ) shows representative luciferase images of mice receiving wtAAV8.CB7.ffluc and N499Q capsid mutant vectors on day 14 after injection. ( Figure 7C ) Luciferase expression was measured by luciferase imaging from C57BL / 6 mice injected intravenously with wt AAV8 vector or mutant vector (n=3 or 4) on day 14 of the study period and reported as total flux units. All data are expressed as mean + standard deviation.

[0030] Figure 8A and Figure 8B Shows the results of in vitro analysis of the effect of gene deamidation on vector performance. ( Figure 8A ) The titers of wtAAV8 vectors and gene deamidation mutant vectors were generated by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). The titers are reported relative to the wtAAV8 control. Highly deamidated NG sites (patterned bars), lowly deamidated sites (white bars), and highly variable sites (black bars) are presented using wtAAV8 and a negative control. ( Figure 8B ) Transduction efficiency of mutant AAV8 vectors producing firefly luciferase reported relative to a wt AAV8 control. Transduction efficiency was measured as luminescence units per GC added to HUH7 cells and was determined by transduction with crude vector at multiple dilutions. Transduction efficiency data were normalized to the wt reference. All data are presented as mean ± standard deviation.

[0031] Figures 9A-9D demonstrated that loss of carrier activity over time is associated with progressive deamidation. Figure 9A ) Vector production over a time course of triple-transfected HEK 293 cells (DNAseI-resistant genomic copies, GC) to produce AAV8 vectors packaging a luciferase reporter gene. GC levels were normalized to the maximum observed value. ( Figure 9B ) Huh7 cells were transduced with purified time-course vectors. Multiple dilutions of purified time-course vector samples were used as Figure 8B Transduction efficiency was measured as in (luminescence units per GC added to target cells) as in

[15] . Error bars represent the standard deviation of at least 10 technical replicates for each sampling time. AAV8 NG sites of vectors collected 1, 2, and 5 days after transfection ( Figure 9C ) and non-NG sites ( Figure 9D ) deamidation.

[0032] Figures 10A-10D The effect of stabilizing asparagine on the carrier performance is demonstrated. Figure 10A Shown are the titers of wtAAV8 vector and +1 position mutant vector generated by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). Titers are reported relative to wtAAV8 control. Figure 10B The transduction efficiency of mutant AAV8 vectors producing firefly luciferase is reported relative to that of wtAAV8 control. Figure 8B Measure transduction efficiency as in . Run a two-sample t test ( * p<0.005) to determine the significance between the transduction efficiency of wtAAV8 and the mutants G264A / G515A and G264A / G541A. Figure 10C Luciferase expression in liver regions from C57BL / 6 mice injected intravenously with wtAAV8 vector or mutant vectors (n=3 to 5) on day 14 of the study period, as measured by luciferase imaging, is shown and reported in total flux units. Figure 10D Shown are the titers and transduction efficiencies of multi-site AAV8 mutant vectors producing firefly luciferase reported relative to wtAAV8 controls. All data are presented as mean ± standard deviation.

[0033] Figures 11A-11C : Analysis of asparagine and glutamine deamidation in AAV9 capsid protein. Figure 11A 1e11 GC of wtAAV9 was analyzed by 2D gel electrophoresis and stained with Sypro Ruby. Protein markers: A = VP1; B = VP2; C = VP3, D = hen egg white conalbumin marker, E = turbouclease marker. Isoelectric focusing was performed at a pI range of 6-10. Figure 11B) shows the percentage of deamidation at specific asparagine and glutamine residues of interest for AAV9 tryptic peptides purified by different methods. Bars indicating deamidation at asparagine residues with N+1 glycine are cross-hatched. Residues determined to be at least 2% deamidated in at least one of the preparations analyzed are included. Data are expressed as mean ± standard deviation. ( Figure 11C ) The isoaspartic acid model of N512 is shown in the 2FoFc electron density map generated by unbiased refinement of the AAV9 crystal structure (PDB ID: 3UX1). The arrow indicates the electron density corresponding to the R group of residue N512.

[0034] Figures 11D-11F : Determination of factors affecting AAV9 capsid deamidation. Figure 11D ) Incubate the two AAV9 preparations at 70°C for three or seven days, or ( Figure 11F ) were exposed to pH 2 or pH 10 for seven days to identify possible sources of deamidation that are not intrinsic to AAV capsid formation. Data are presented as mean ± standard deviation. ( Figure 11F ) Using B1 antibody (reacts with denatured capsid) Figure 11D Dot blots of the vectors were processed as such to assess capsid structural integrity.

[0035] Figure 11G and Figure 11H In vitro analysis of the effect of AAV9 gene deamidation on vector performance is presented. Figure 11G ) The titers of wtAAV9 vectors and gene deamidation mutant vectors were generated by small-scale triple transfection in 293 cells, as measured by quantitative PCR (qPCR). The titers are reported relative to the wtAAV9 control. Highly deamidated NG sites (patterned bars), lowly deamidated sites (white bars), and highly variable sites (black bars) are presented using wtAAV8 and a negative control. ( Figure 11H ) Transduction efficiency of mutant AAV9 vectors producing firefly luciferase is reported relative to wtAAV9 control. All data are expressed as mean ± standard deviation.

[0036] Figures 11I-11K Shown is the in vitro efficacy of AAV9 vectors over time. ( Figure 11I ) Vector production over a time course of triple-transfected HEK293 cells (DNAseI-resistant genome copies, GC) to produce AAV9 vectors packaging a luciferase reporter gene. GC levels were normalized to the maximum observed value. ( Figure 11J ) Huh7 cells were transduced with the crude time-course vector. ( Figure 11K) shows the transduction efficiency of vector collected 1 day after transfection versus 5 days after transfection for crude vector samples and purified vector samples. Transduction efficiency is expressed as luciferase activity / GC, normalized to the value on day 1.

[0037] Figure 12A Provided is an alignment of the amino acid sequences of AAV5.5.9 [SEQ ID NO: 10] (sometimes also referred to as AAVG5), AAV9 [SEQ ID NO: 4], and AAVPHP.B [SEQ ID NO: 12] prepared using Clustal Ω 1.2.2 and its default parameters for alignment. Figures 12B-12E An alignment of the nucleotide sequences of AAV5.5.9 [SEQ ID NO:9], PHP.B [SEQ ID NO:11], AAV9 [SEQ ID NO:3], and AAVhu68 [SEQ ID NO:14] is provided.

[0038] Figure 13A Provided is an alignment of the amino acid sequences of AAV8 triple mutant (AAV8T) [SEQ ID NO: 16], AAV8.AR2.08 [SEQ ID NO: 18] (also sometimes referred to as AAVG3 or AR2 or AAV.AR2), and AAV8 [SEQ ID NO: 20], prepared using ClustalΩ 1.2.2 and its default parameters for alignment. Figures 13B-13D An alignment of the nucleotide sequences of AAV8 triple mutant [SEQ ID NO: 15], AAV8.AR2.08 [SEQ ID NO: 17], and AAV8 [SEQ ID NO: 19] is provided.

[0039] Figure 14A Provided is an alignment of the amino acid sequences of AAVrh79 [SEQ ID NO: 2] (sometimes also referred to as AAVG2), AAVrh10 [SEQ ID NO: 24], and AAVhu37 [SEQ ID NO: 22] using ClustalΩ 1.2.2 and its default parameters for alignment. Figures 14B-14D An alignment of the nucleotide sequences of AAVrh79 [SEQ ID NO: 1], AAVrh10 [SEQ ID NO: 22], and AAVhu37 [SEQ ID NO: 21] is provided.

[0040] Figure 15A and 15B Production productivity of AAV8 triplex, AAVhu68, AAV9, AAV9, and AAVrh79 in small-scale or large-scale formulations of reference vectors is shown.

[0041] Figure 16 Provided Figure 15B The purity of the resulting large-scale preparations.

[0042] Figures 17A to 17D The vector expressing firefly luciferase was used to convert 3×10 11 Luciferase expression in liver and muscle tissues after intramuscular (IM) administration of GC / mouse into the gastrocnemius muscle of male C57BL / 6 mice (n=5 / group). Figure 17E Shows that 10 13 Expression of AVV8 triple, AAVhu68, AAV9, AAV8, and AAVrh79 vectors after GC / kg AAVrh79 intramuscular injection into male and female cynomolgus monkeys. Figure 17F The vector containing the transgene (3x10 10 or 3x10 11 Expression of secreted transgene (201Ig IA) after intramuscular injection of 201Ig IA (n=5 / group) into the gastrocnemius muscle of male RAG KO mice (n=5 / group).

[0043] Figure 18A The plasmids used for barcoding experiments in Example 5 are shown. Figure 18B The amount of each AAV barcode variant injected into six black mice is shown. The animals were sacrificed, tissue samples were collected, and DNA was isolated from each of these animals. Figure 18C Total vector distribution for three animals is shown in . Figure 18D The actual frequency of the injected vector mixture is shown in versus the theoretical frequency.

[0044] Figures 19A-20C The results of the barcode biodistribution experiment of Example 5 are shown. The frequency of individual barcodes in samples for genomic and cDNA were analyzed for individual tissue samples versus the frequency of individual barcodes in the injection mixture. Figure 19A , 19B); heart ( Figure 19C and Figure 19D ) and liver( Figure 19E and Figure 19F ) results. Figures 20A-20C The fold changes compared to the theoretical frequencies are shown in .

[0045] Figure 21 and 22 The biodistribution of AAV8.AR2.08 in mice compared to AAV8 is shown. The results indicate that AAV8.AR2.08 has higher liver specificity than AAV8.

[0046] Figure 23The titer and yield related to manufacturability of AAV8 vs. AAV8.AR2.08 vs. AAVrh79 were compared.

[0047] Figure 24 Shown is the biodistribution of AAV8.AR2.08 in tissues (leftmost bar) compared to AAV8 (middle and right bars).

[0048] Figures 25-28 Shown are the results following administration of AAV vectors to non-human primates. Figure 25 Details of the vectors and animals used for the studies are provided. Figure 26 quantifies the levels of GC and GFP detected in livers from animals receiving AAV8, AAVrh79, or AAV8.AR2.08 vectors. Figure 27 summarizes the levels of GFP expression in HNP livers. Figure 28 Shown are the levels of vector detected in tissues from HNPs administered with AAV8, AAVrh79, or AAV8.AR2.08 vectors.

[0049] Figure 29 Shown is the biodistribution of AAVrh79 vector detected in various tissues. DETAILED DESCRIPTION

[0050] Provided herein is a recombinant adeno-associated virus (rAAV) and compositions containing the same, having sequence and charge heterogeneity in each of the three capsid protein populations VP1, VP2, and VP3 present within the capsid of the recombinant AAV. Provided herein are novel rAAVs and methods for reducing deamidation and optionally other capsid monomer modifications. Further provided herein are modified rAAVs with reduced modifications that can be used to provide rAAVs with capsids that maintain greater stability, potency, and / or purity.

[0051] "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle containing two elements, the AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid. Unless otherwise indicated, this term can be used interchangeably with the phrase "rAAV vector". rAAV is a "replication-defective virus" or "viral vector" because it lacks any functional AAVrep gene or functional AAVcap gene and cannot produce offspring. In certain embodiments, the only AAV sequences are the AAV inverted terminal repeats (ITRs), which are typically located at the 5' and 3' extreme ends of the vector genome to allow genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0052] As used herein, " vector genome " refers to the nucleic acid sequence packaged inside the rAAV capsid that forms viral particles. This nucleic acid sequence contains AAV inverted terminal repeats (ITR). In the examples herein, the vector genome contains at least AAV 5'ITR, one or more coding sequences and AAV 3'ITR from 5' to 3'. It is possible to select the ITR from AAV2, be different from the capsid source or the AAV except the full-length ITR. In certain embodiments, ITR is from the AAV source identical with the AAV that provides rep function during production or trans-supplementation AAV. Further, other ITR can be used. Further, the vector genome contains the regulatory sequence that guides the expression of the gene product. The suitable components of the vector genome are discussed in more detail herein.

[0053] rAAV consists of an AAV capsid and a vector genome. The AAV capsid is an assembly of a heterogeneous population of vp1, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein, when used to refer to vp capsid proteins, the term "heterologous" or any grammatical variation thereof refers to a population composed of non-identical elements, such as vp1, vp2, or vp3 monomers (proteins) having different modified amino acid sequences.

[0054] As used herein, the term "heterogeneous population" used in conjunction with the vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications from predicted amino acid residues. These subpopulations contain at least some deamidated asparagine (N or Asn) residues. For example, certain subpopulations include at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further include other deamidated amino acids, wherein the deamidation results in amino acid changes and other optional modifications.

[0055] As used herein, unless otherwise specified, a "subpopulation" of vp proteins refers to a group of vp proteins that has at least one defined characteristic and consists of at least one member of a group to fewer than all members of a reference group. For example, a "subpopulation" of vp1 proteins can be at least one (1) vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid, unless otherwise specified. A "subpopulation" of vp3 proteins can be one (1) vp3 protein to fewer than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vp1 proteins can be a subpopulation of vp proteins; vp2 proteins can be a different, separate subpopulation of vp proteins, and vp3 is yet another subpopulation of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can contain subpopulations with different modifications, such as at least one, two, three, or four highly deamidated asparagines, such as at asparagine-glycine pairs.

[0056] Unless otherwise indicated, highly deamidated refers to a reference amino acid position that is at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or up to about 100% deamidated compared to the predicted amino acid sequence at the reference amino acid position (e.g., at least 80% of the asparagine at amino acid 57 of SEQ ID NO: 2 may be deamidated based on the total vp1 protein or may be deamidated based on the total vp1, vp2, and vp3 proteins). Such percentages can be determined using 2D gels, mass spectrometry, or other suitable techniques.

[0057] Without wishing to be bound by theory, deamidation of at least highly deamidated residues in the vp protein in the AAV capsid is believed to be primarily non-enzymatic in nature, resulting from functional groups within the capsid protein that deamidate selected asparagine residues and, to a lesser extent, glutamine residues. The efficient capsid assembly of the majority of deamidated vp1 proteins suggests that these events occur after capsid assembly, or that deamidation in individual monomers (vp1, vp2, or vp3) is well tolerated structurally and does not largely affect assembly kinetics. Extensive deamidation in the VP1 unique (VP1-u) region (approximately aa 1-137), which is generally considered to be localized internally prior to cell entry, suggests that VP deamidation may occur prior to capsid assembly.

[0058] Without wishing to be bound by theory, the deamidation of N can occur by a nucleophilic attack on the side chain amide carbon atom of Asn by the backbone nitrogen atom of its C-terminal residue. It is believed that an intermediate closed-loop succinimide residue is formed. The succinimide residue is then rapidly hydrolyzed to produce the final product aspartic acid (Asp) or isoaspartic acid (IsoAsp). Therefore, in certain embodiments, the deamidation of asparagine (N or Asn) produces Asp or IsoAsp, which can be mutually converted by a succinimide intermediate, such as shown below.

[0059]

[0060] As provided herein, each deamidated N in VP1, VP2, or VP3 can independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartic acid and / or an interconversion blend of Asp and isoAsp, or a combination thereof. Any suitable ratio of α- and isoaspartic acid can be present. For example, in certain embodiments, the ratio can be 10:1 to 1:10 aspartic acid:isoaspartic acid, about 50:50 aspartic acid:isoaspartic acid, or about 1:3 aspartic acid:isoaspartic acid, or another selected ratio.

[0061] In certain embodiments, one or more glutamines (Q) can be deamidated to glutamic acid (Glu), i.e., α-glutamic acid, γ-glutamic acid (Glu), or a blend of α- and γ-glutamic acid, which can be interconverted via a common glutarimide intermediate. α- and γ-glutamic acid can be present in any suitable ratio. For example, in certain embodiments, the ratio can be 10:1 to 1:10 α:γ, about 50:50 α:γ, or about 1:3 α:γ, or another selected ratio.

[0062]

[0063] Thus, the rAAV capsids comprising the vp1, vp2, and / or vp3 proteins comprise a subpopulation of deamidated amino acids, including at least one subpopulation comprising at least one highly deamidated asparagine. Additionally, other modifications may comprise isomerization, particularly at selected aspartic acid (D or Asp) residue positions. In still other embodiments, the modification may comprise amidation at the Asp position.

[0064] In certain embodiments, the AAV capsid contains a subpopulation 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, wherein at least 1% to 10%, at least 10% to 25%, at least 25% to 50%, at least 50% to 70%, at least 70% to 100%, at least 75% to 100%, at least 80% to 100%, or at least 90% to 100% of the vp1, vp2, and vp3 are deamidated compared to the encoded amino acid sequence of the vp protein. The majority of these can be N residues. However, Q residues can also be deamidated.

[0065] As used herein, "encoded amino acid sequence" refers to the predicted amino acid based on translation of the known DNA codons of the referenced nucleic acid sequence that is translated into amino acids. The following table shows DNA codons and twenty common amino acids, showing single-letter codes (SLC) and three-letter codes (3LC), respectively.

[0066]

[0067]

[0068] In certain embodiments, the rAAV has an AAV capsid containing vp1, vp2, and vp3 proteins having a subpopulation comprising a combination of two, three, four, five, or more deamidated residues at the positions listed in the Tables provided herein and incorporated by reference herein.

[0069] Deamidation in rAAV can be determined using 2D gel electrophoresis and / or mass spectrometry and / or protein modeling techniques. Online chromatography can be performed using an Acclaim PepMap column and a Thermo UltiMate 3000RSLC system (Thermo Fisher Scientific) coupled to a Q Exactive HF and NanoFlex source (Thermo Fisher Scientific). MS data were acquired using a data-dependent top 20 method for the Q Exactive HF that dynamically selects the most abundant precursor ions that have not yet been sequenced from the survey scan (200-2000 m / z). Sequencing was performed by high-energy collisional fragmentation with a target value of 1e5 ions determined by predictive automatic gain control, and precursor separation was performed with a window of 4 m / z. Survey scans were acquired at m / z 200 with a resolution of 120,000. The resolution of the HCD spectrum can be set to 30,000 at m / z 200, with a maximum ion injection time of 50 milliseconds and a normalized collision energy of 30. The S-lens RF level can be set to 50 to achieve optimal transmission in the m / z zone occupied by the digestive peptide. Precursor ions with a single, unassigned, or six and higher charge states can be excluded from the fragment selection. BioPharmaFinder 1.0 software (Thermo Fisher Scientific) can be used to analyze the data obtained. For peptide mapping, a single entry protein FASTA database is used to search, with carbamidomethylation being set to a fixed modification; and oxidation, deamidation, and phosphorylation being set to a variable modification, 10ppm mass accuracy, high protease specificity, and a confidence level of 0.8 MS / MS spectrum. The example of a suitable protease can include, for example, trypsin or chymotrypsin. The mass spectrometric identification of the deamidated peptide is relatively simple because deamidation adds +0.984Da (the mass difference between the OH group and the NH2 group) to the mass of the intact molecule. The deamidation percentage 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. Taking into account the number of possible deamidation sites, isobaric species deamidated at different sites can co-migrate in a single peak. Therefore, fragment ions derived from peptides with multiple potential deamidation sites can be used to locate or distinguish multiple deamidation sites. In these cases, the relative intensity in 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 of all isomeric species is the same and is independent at the deamidation site. It will be appreciated by those skilled in the art that various modifications of these illustrative methods can be used.For example, suitable mass spectrometers can include, for example, quadrupole time-of-flight mass spectrometers (QTOF), such as Waters Xevo or Agilent 6530, or Orbitrap instruments, such as Orbitrap Fusion or Orbitrap Velos (Thermo Fisher Scientific). Suitable liquid chromatography systems include, for example, Acquity UPLC systems or Agilent systems (1100 or 1200 series) from Waters (Waters). Suitable data analysis software can include, for example, MassLynx (Waters), Pinpoint and Petfinder (Thermo Fisher Scientific), Mascot (Matrix Science), Peaks DB (Bioinformatics Solutions). Other technologies can be described in, for example, X. Jin et al., " Human Gene Therapy Methods " published online on June 16, 2017, Vol. 28, No. 5, pp. 255-267.

[0070] In addition to deamidation, other modifications that do not result in the conversion of one amino acid into a different amino acid residue may occur. Such modifications may include acetylated residues, isomerization, phosphorylation or oxidation.

[0071] Regulation of deamidation: In certain embodiments, AAV is modified to change the glycine in the asparagine-glycine pair to reduce deamidation. In other embodiments, asparagine is changed to a different amino acid, such as glutamine, which is deamidated at a slower rate; or changed to an amino acid lacking an amide group (e.g., glutamine and asparagine containing an amide group); and / or changed to an amino acid lacking an amide group (e.g., lysine, arginine, and histidine containing 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 can be in one, two, or three asparagine-glycine pairs in the asparagine-glycine pairs present in the encoded AAV amino acid sequence. In certain embodiments, no such modifications are made in all four asparagine-glycine pairs. Therefore, a method for reducing the deamidation of AAV and / or engineered AAV variants with a lower deamidation rate. Additionally or alternatively, one or more other amide amino acids can be changed to non-amide amino acids to reduce deamidation of AAV. In certain embodiments, the mutant AAV capsids described herein contain mutations in asparagine-glycine pairs such that glycine becomes alanine or serine. The mutant AAV capsids can contain one, two, or three mutants, wherein the reference AAV naturally contains four NG pairs. In certain embodiments, the AAV capsids can contain one, two, three, or four such mutants, wherein the reference AAV naturally contains five NG pairs. In certain embodiments, the mutant AAV capsids contain only a single mutation in the NG pair. In certain embodiments, the mutant AAV capsids contain mutations in two different NG pairs. In certain embodiments, the mutant AAV capsids contain mutations in two different NG pairs that are located in structurally separate positions in the AAV capsid. In certain embodiments, the mutations are not in the VP1 unique region. In certain embodiments, one of the mutations is not in the VP1 unique region. Optionally, the mutant AAV capsid does not contain modifications in the NG pair, but contains mutations to minimize or eliminate deamidation in one or more asparagine or glutamine located on the outside of the NG pair.

[0072] In certain embodiments, a method for increasing the effectiveness of an rAAV vector is provided, the method comprising engineering an AAV capsid, which eliminates one or more NGs in the NG in the wild-type AAV capsid. In certain embodiments, the coding sequence of the "G" of "NG" is engineered to encode another amino acid. In certain examples below, "S" or "A" is substituted. However, other suitable amino acid coding sequences can be selected. See, for example, the table below, wherein the coding sequence for at least one of the following positions is modified based on the numbering of AAV8: N57+1, N263+1, N385+1, N514+1, N540+1, or as shown in the table below. In certain embodiments, the AAV8 mutant avoids changing the NG pair at positions N57, N94, N263, N305, Q467, N479 and / or N653. In certain embodiments, other AAVs avoid mutations at corresponding N positions as determined based on comparison with AAV8 using AAV8 numbering as a reference.

[0073] These amino acid modifications can be performed by conventional genetic engineering techniques. For example, a nucleic acid sequence containing a modified AAVvp codon can be generated in which one to three codons encoding glycine in the arginine-glycine pair are modified to encode an amino acid other than glycine. In certain embodiments, a nucleic acid sequence containing a modified arginine codon can be engineered at one to three arginine-glycine pairs in the arginine-glycine pair so that the modified codon encodes an amino acid other than arginine. Each modified codon can encode a different amino acid. Alternatively, one or more codons in the altered codons can encode the same amino acid. In certain embodiments, these modified AAVrh79, AAV8.AR2.08 or AAV5.5.9 nucleic acid sequences can be used to generate mutant rAAVs having a capsid with a lower degree of deamidation than the native AAVrh79, AAV8.AR2.08 or AAV5.5.9 capsid. Such mutant rAAV may have reduced immunogenicity and / or increased stability upon storage, particularly when stored in suspension form.

[0074] Also provided herein is a nucleotide sequence encoding the deamidated AAV capsid with reduction. Designing the nucleotide sequence encoding this AAV capsid is within the technical scope of this area, including DNA (genome or cDNA) or RNA (e.g., mRNA). Such nucleotide sequences that can be codon-optimized to be expressed in a selected system (i.e., cell type) can be designed by various methods. This optimization can be performed using online available methods (e.g., GeneArt), disclosed methods, or a company (e.g., DNA2.0) (Menlo Park, California) that provides codon optimization services. For example, a codon optimization method is described in International Patent Publication No. WO 2015 / 012924, which is incorporated herein by reference in its entirety. Also referring to, for example, U.S. Patent Publication No. 2014 / 0032186 and U.S. Patent Publication No. 2006 / 0136184. Suitable for modifying the full length of the open reading frame (ORF) of the product. However, in some embodiments, only the fragment of the ORF can be changed. By using a method in these methods, frequency can be applied to any given peptide sequence, and produce the nucleic acid fragment of the codon-optimized coding region that polypeptide is encoded.Many options can be used for codon being actually changed or can be used for synthesizing the codon-optimized coding region of design as described herein.This type of change or synthesis can use the standard and conventional molecular biology operation that those of ordinary skill in the art are well known to carry out.In one method, a series of complementary oligonucleotide pairs of the length that is 80-90 Nucleotide and span the sequence of hope are synthesized separately by standard method.These oligonucleotide pairs are to being synthesized so that when annealing, they form the double-stranded fragment of 80-90 base pair, and these double-stranded fragments contain sticky end, and for example, each oligonucleotide in this pair is synthesized to extend beyond 3,4,5,6,7,8,9,10 or more bases in the region that another oligonucleotide is complementary to this pair.Every pair of the single-stranded end of oligonucleotide is designed to anneal with the single-stranded end of another pair of oligonucleotide. These oligonucleotide pairs are allowed to anneal, and then about five to six of these double-stranded fragments are allowed to anneal together via the sticky single-stranded ends, and then they are ligated together and cloned into a standard bacterial cloning vector, such as the one available from Invitrogen Corporation, Carlsbad, Calif. Vector. Then the construct is sequenced by standard methods. Prepare several constructs in these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments (i.e., fragments of about 500 base pairs) that are linked together so that the sequence of the whole hope is represented with a series of plasmid constructs. Then the insert of these plasmids is cut with appropriate restriction enzymes and linked together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Other methods will be clear to the technician immediately. In addition, gene synthesis is easy to commercially available.

[0075] In certain embodiments, AAV capsids are provided that have a heterogeneous population of AAV capsid isotypes (i.e., VP1, VP2, VP3) containing multiple highly deamidated "NG" positions. In certain embodiments, the highly deamidated positions are among the positions identified below with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that the reference "NG" is ablated and a mutant "NG" is engineered into another position.

[0076] In certain embodiments, the mixed rAAV population is produced by a production system using a single AAV capsid nucleic acid sequence that encodes the predicted AAV VP1 amino acid sequence of one AAV type. However, the production and manufacturing processes provide a heterogeneous population of capsid proteins as described above.

[0077] In certain embodiments, novel isolated AAVrh79 capsids are 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.

[0078] In certain embodiments, the rAAV comprises an AAVrh79 capsid. The AAVrh79 capsid comprises a heterogeneous population of AAVrh79 vp1, AAVrh79 vp2, and AAVrh79 vp3 proteins. In one embodiment, the AAVrh79 capsid is produced by expressing a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2 from 1 to 738. Optionally, the sequence co-expresses a vp3 protein from a nucleic acid sequence that does not include the vp1 unique region (approximately aa 1 to 137) or the vp2 unique region (approximately aa 1 to 203), a vp1 protein produced from SEQ ID NO: 1, or a vp1 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2 from 1 to 738 that is at least 70% identical to SEQ ID NO: 1. 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 about amino acids 138 to 738 of SEQ ID NO:2, a vp2 protein is produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO:1, or a vp2 protein is produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO:2 that is at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO:1, an AAVrh79 vp3 protein is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO:2, a vp3 protein is produced from a sequence comprising at least nucleotides 610 to 2214 of SEQ ID NO:1, or a nucleic acid sequence encoding at least nucleotides 610 to 2214 of SEQ ID NO:1 that is at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO:1. The VP3 protein is produced by a nucleic acid sequence encoding at least about amino acids 204 to 738 of the predicted amino acid sequence of NO:2.

[0079] In certain embodiments, the AAVrh79 capsid comprises: a heterogeneous population of vp1 proteins, which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2; a heterogeneous population of vp2 proteins, which are the product of a nucleic acid sequence encoding at least about amino acids 138 to 738 of SEQ ID NO:2; and a heterogeneous population of vp3 proteins, which are the product of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO:2.

[0080] The AAVrh79 vp1, vp2, and vp3 proteins contain a subpopulation of amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs of SEQ ID NO:2, and optionally further comprise a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. Relative to the numbering of SEQ ID NO:2, high levels of deamidation were observed at the N-G pairs N57, N263, N385, and / or N514. Deamidation was also observed at other residues, as shown in the following table and examples. In certain embodiments, AAVrh79 may have other deamidated residues, e.g., typically less than 10%, and / or may have other modifications including methylation (e.g., about R487) (typically less than 5%, more typically less than 1% at a given residue), isomerization (e.g., at D97) (typically less than 5%, more typically less than 1% at a given residue), phosphorylation (e.g., where present, ranging from about 10% to about 60%), or about 10% to about 30%, or about 20% to about 60%) (e.g., at one or more of S149, about S153, about S474, about T570, about S665) or oxidized (e.g., at one or more of W248, W307, W307, M405, M437, M473, W480, W480, W505, M526, M544, M561, W621, M637, and / or W697). Optionally, W can be oxidized to kynurenine.

[0081] Table A - AAVrh79 deamidation

[0082]

[0083] In certain embodiments, the AAVrh79 capsid is modified at one or more of the positions identified in the table above, within the ranges provided below as determined using mass spectrometry using trypsin. In certain embodiments, one or more of the following positions or the glycine following the N are modified as described herein. The residue numbers are based on the AAVrh79 sequence provided herein. See SEQ ID NO: 2.

[0084] In certain embodiments, a nucleic acid sequence encoding the AAVrh79 vp1 capsid protein is provided in SEQ ID NO: 1. In other embodiments, a nucleic acid sequence having 70% to 99.9% identity to SEQ ID NO: 1 can 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% identical, at least 90% identical, at least 95% identical, 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 can be selected for use in generating rAAV capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 1 or a sequence encoding SEQ ID NO: 2 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: 1. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 1, or a sequence encoding the vp2 capsid protein (about aa 138 to 738) of SEQ ID NO: 2 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%, or at least 99% identical to about nt 412 to about nt 2214 of SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of about nt 610 to about nt 2214 of SEQ ID NO: 1, or a sequence encoding the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 2 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%, or at least 99% identical to nt SEQ ID NO: 1.

[0085] The present invention also encompasses nucleic acid sequences encoding mutant AAVrh79 in which one or more residues have been altered to reduce deamidation or other modifications identified herein. Such nucleic acid sequences can be used to generate mutant rAAVrh79 capsids.

[0086] In certain embodiments, novel AAV8.AR2.08 capsids are 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, a recombinant adeno-associated virus (rAAV) has an AAV8.AR2.08 capsid. Figure 13A An alignment of the amino acid sequences of AAV8T, AAV8.AR2.08, and AAV8 is provided in . Figures 13B-13DAn alignment of the nucleic acid sequences of AAV8T, AAV8.AR2.08, and AAV8 is provided in .

[0087] In certain embodiments, the AAV8.AR2.08 capsid comprises an AAV8.AR2.08 capsid protein comprising: an AAV8.AR2.08 vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 18, a vp1 protein produced from SEQ ID NO: 17, or a vp1 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 738 of SEQ ID NO: 18 that is at least 70% identical to SEQ ID NO: 17, an AAV8.AR2.08 vp2 protein produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 738 of SEQ ID NO: 18, a vp2 protein produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO: 17, or a vp2 protein produced from a nucleic acid sequence encoding at least nucleotides 412 to 2214 of SEQ ID NO: 17 that is at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO: 17. NO: 18, the AAV8.AR2.08 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO: 18, the AAV8.AR2.08 vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 18, the vp3 protein produced from a sequence comprising at least nucleotides 610 to 2214 of SEQ ID NO: 17, or the vp3 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO: 18 that is at least 70% identical to at least nucleotides 610 to 2214 of SEQ ID NO: 17.

[0088] Additionally or alternatively, the AAV8.AR2.08 capsid comprises: a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 138 to 738 of SEQ ID NO: 18; and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 204 to 738 of SEQ ID NO: 18, wherein: the vp1 proteins, the vp2 proteins, and the vp3 proteins contain a subpopulation having amino acid modifications that include at least two highly deamidated asparagines (N) of the asparagine-glycine pairs in SEQ ID NO: 18, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. AAV8.AR2.08 is characterized by residues having a high degree of deamidation 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 positions in the following tables and detailed tables of the present application show deamidation that has been observed in the AAV8.AR2.08 capsid.

[0089] In certain embodiments, the AAV8.AR2.08 capsid is modified at one or more of the following positions within the ranges provided below as determined using mass spectrometry using trypsin. In certain embodiments, the glycine following one or more of the following positions or N is modified as described herein. For example, in certain embodiments, G can be modified to S or A, for example, at positions 58, 264, 386, 515, or 541. When NG57 / 58 is changed to NS57 / 58 or NA57 / 58, a significant decrease in deamidation is observed. However, in certain embodiments, when NG is changed to NS or NA, an increase in deamidation is observed. In certain embodiments, the N of the NG pair is modified to Q while retaining the G. In certain embodiments, both amino acids of the NG pair are modified. In certain embodiments, N385Q causes a significant decrease in deamidation at the position. In certain embodiments, N499Q causes a significant increase in deamidation at the position.

[0090] In addition to deamidation, other modifications may include isomerization (e.g., at one or more of D442 and / or D584) (1-15%), phosphorylation (e.g., at one or more of about S149, about T417, about T454, about T493, S600, and / or about T663), and / or oxidation (e.g., at one or more of about W22, about M204, about M212, W248, W307, M405, M437, M473, W480, W505,

[0091] M526, M561, M607, about one or more of W609, W621, M637, W697). Other positions may have such or other modifications (e.g., acetylation or additional deamidation).

[0092] Table B - AAV8.AR2.08 deamidation

[0093]

[0094] 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 having 70% to 99.9% identity to SEQ ID NO: 17 can 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% identical, at least 90% identical, at least 95% identical, 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 can be selected for use in generating rAAV8.AR2.08 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 17, or a sequence encoding SEQ ID NO: 18 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. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 17, or a sequence encoding the vp2 capsid protein (approximately aa 138 to 738) of SEQ ID NO: 18 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%, or at least 99% identical to about nt 412 to about nt 2214 of SEQ ID NO: 17. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of about nt 607 to about nt 2214 of SEQ ID NO: 17, or a sequence encoding the vp3 capsid protein (about aa 204 to 738) of SEQ ID NO: 18 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%, or at least 99% identical to nt SEQ ID NO: 17.

[0095] The present invention also encompasses nucleic acid sequences encoding mutant AAV8.2.08 in which one or more residues have been altered to reduce deamidation or other modifications identified herein. Such nucleic acid sequences can be used to generate mutant rAAV8.2.08.

[0096] In certain embodiments, novel AAV5.5.9 capsids are 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. Figure 12A An alignment of the amino acid sequences of AAV5.5.9, AAV9, and AAVPHP.B is shown in FIG. Figures 12B-12EAn alignment of the nucleic acid sequences of AAV5.5.9, AAV9, and AAVPHP.B is shown in FIG. In one embodiment, the recombinant adeno-associated virus (rAAV) has an AAV5.5.9 capsid comprising: AAV5.5.9 capsid proteins comprising: a heterogeneous population of: AAV5.5.9 vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 726 of SEQ ID NO: 10, vp1 proteins produced from SEQ ID NO: 9, or vp1 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 726 of SEQ ID NO: 1 that is at least 70% identical to SEQ ID NO: 9; and a heterogeneous population of: AAV5.5.9 vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 137 to 726 of SEQ ID NO: 10, vp2 proteins produced from a sequence comprising at least nucleotides 409 to 2178 of SEQ ID NO: 9, or vp2 proteins produced from a nucleic acid sequence encoding at least 70% identical to SEQ ID NO: 9. NO:9, vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 577 to 2178 of SEQ ID NO:9, encoding the predicted amino acid sequence of at least about amino acids 137 to 726 of SEQ ID NO:10; and the following heterogeneous populations: AAV5.5.9 vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 193 to 726 of SEQ ID NO:10, vp3 protein produced from a sequence including at least nucleotides 577 to 2178 of SEQ ID NO:9, or vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 577 to 2178 of SEQ ID NO:9, encoding the predicted amino acid sequence of at least about amino acids 193 to 726 of SEQ ID NO:10.

[0097] Additionally or alternatively, the AAV5.5.9 capsid comprises: a heterogeneous population of vp1 proteins, which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10; a heterogeneous population of vp2 proteins, which are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 137 to 726 of SEQ ID NO: 10; and a heterogeneous population of vp3 proteins, which are the product of a nucleic acid sequence encoding at least amino acids 193 to 726 of SEQ ID NO: 10, wherein: the vp1 proteins, the vp2 proteins, and the vp3 proteins contain a subpopulation having amino acid modifications, wherein the amino acid modifications include at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO: 10, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change.

[0098] Table C - AAV5.5.9 deamidation

[0099]

[0100]

[0101] 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 having 70% to 99.9% identity to SEQ ID NO:9 can 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% identical, at least 90% identical, at least 95% identical, 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 can be selected for use in generating rAAV5.5.9 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:10 or a sequence encoding SEQ ID NO:10 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:9. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 9, or a sequence encoding the vp2 capsid protein (approximately aa 137 to 726) of SEQ ID NO: 10 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%, or at least 99% identical to about nt 409 to about nt 2178 of SEQ ID NO: 9. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of about nt 577 to about nt 2178 of SEQ ID NO: 9, or a sequence encoding the vp3 capsid protein (approximately aa 193 to 726) of SEQ ID NO: 10 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%, or at least 99% identical to nt 577 to 2178 of SEQ ID NO: 9.

[0102] The present invention also encompasses nucleic acid sequences encoding mutant AAV5.5.9 in which one or more residues have been altered to reduce deamidation or other modifications identified herein. Such nucleic acid sequences can be used to generate mutant rAAV5.5.9.

[0103] I. rAAV vector

[0104] As indicated above, novel AAV sequences and proteins can be used to generate rAAV and can also be used in recombinant AAV vectors, which can be antisense delivery vectors, gene therapy vectors, or vaccine vectors. In addition, the engineered AAV capsids described herein can be used to engineer rAAV vectors to deliver a variety of suitable nucleic acid molecules to target cells and tissues.

[0105] The genomic sequence packaged into the AAV capsid and delivered to the host cell is generally composed of at least a transgene and its regulatory sequences and AAV inverted terminal repeats (ITRs). Both single-stranded AAV and self-complementary (sc) AAV are encompassed within rAAV. A transgene is a nucleic acid coding sequence that is heterogeneous to the vector sequence and that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid coding sequence is operably linked to a regulatory component in a manner that allows the transgene to be transcribed, translated and / or expressed in the cells of the target tissue.

[0106] The AAV sequence of the vector typically includes cis-acting 5' and 3' inverted terminal repeats (see, for example, BJ Carter, Handbook of Parvoviruses, edited by P. Tijsser, CRC Press, pp. 155-168 (1990)). The length of the ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, although minor modifications to these sequences are permitted to a certain extent. The ability to modify these ITR sequences is within the skill of the art. (See, for example, texts such as Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70: 520-532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing a transgene in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are derived from an AAV different from the AAV that supplies the capsid, thereby generating a pseudotyped vector. In one embodiment, the ITRs are derived from AAV2. A shortened version of the 5' ITR, referred to as ΔITR, has been described in which the D sequence and the terminal resolution site (trs) are deleted. In other embodiments, full-length AAV 5' and 3' ITRs are used. However, ITRs from other AAV sources can be selected. In the case where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0107] In addition to the above-identified major elements of the recombinant AAV vector, the AAV vector also contains the necessary conventional control elements that are operably linked to the transgene in a manner that allows its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include expression control sequences that are contiguous to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0108] Regulatory control elements typically contain a promoter sequence as part of an expression control sequence, for example positioned between a selected 5' ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters [see, for example, WO 2011 / 126808 and WO 2013 / 04943], tissue-specific promoters, or promoters responsive to physiological cues can be used in the vectors described herein. One or more promoters can be selected from different sources, such as human cytomegalovirus (CMV) immediate early enhancer / promoter, SV40 early enhancer / promoter, JC polyoma virus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma 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 metalloproteinase promoter (MPP) and chicken beta-actin promoter. In one embodiment, the promoter is a liver-specific promoter, such as the promoter referred to as LSP exemplified herein.

[0109] In addition to the promoter, the vector may also contain one or more other suitable transcription initiation, termination, and enhancer sequences, efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, such as WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include enhancers suitable for the desired target tissue indication. In one embodiment, the expression cassette includes one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers can be the same or different from each other. For example, the enhancer can include the CMV immediate early enhancer. Such an enhancer can be present in two copies positioned adjacent to each other. Alternatively, the two copies of the enhancer can be separated by one or more sequences. In still another embodiment, the expression cassette further contains an intron, for example, the chicken β-actin intron. Other suitable introns include those known in the art, for example, those described in WO 2011 / 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 can be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which can be engineered upstream of the polyA sequence and downstream of the coding sequence [see, for example, MAZanta-Boussif et al., Gene Therapy (2009) 16:605-619].

[0110] These rAAVs are particularly suitable for gene delivery for therapeutic purposes and for immunity, including inducing protective immunity. Further, the compositions of the present invention can also be used for producing desired gene products in vitro. For in vitro production, the desired product (e.g., protein) can be obtained from the desired culture after rAAV transfection host cells containing molecules encoding the desired product and culturing cell cultures under conditions that allow expression. The expressed product can then be purified and separated as needed. Suitable techniques for transfection, cell culture, purification and separation are known to those skilled in the art.

[0111] Therapeutic transgenic

[0112] The useful products encoded by the transgene include various gene products that replace defective or defective genes, inactivate or "knock out", or "knock down" or reduce the expression of genes that express or deliver gene products with desired therapeutic effects at undesirably high levels. In most embodiments, the therapy will be "somatic cell gene therapy", that is, the gene is transferred to human cells that do not produce sperm or eggs. In certain embodiments, the transgenic expressed protein has a sequence that is a natural human sequence. However, in other embodiments, a synthetic protein is expressed. Such proteins can be used to treat humans, or in other embodiments, are designed to treat animals, including companion animals such as canine or feline groups, or for treating livestock or other animals that come into contact with human populations.

[0113] Examples of suitable gene products can include gene products associated with familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such rare diseases can include 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, Friedrich's ataxia (e.g., ataxin), progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive nonfluent aphasia (PNFA), and semantic dementia), and the like. See, e.g., www.orpha.net / consor / cgi-bin / Disease_Search_List.php; rarediseases.info.nih.gov / diseases.

[0114] Examples of suitable genes can include, for example, hormones and growth and differentiation factors, including but not limited to 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), angiogenin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO) (including, for example, human, canine or feline epo), connective tissue growth factor (CTGF), neurotrophic factors including, for example, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), any of the transforming growth factor alpha superfamily (including TGFα, activin, inhibin), or bone morphogenetic protein (BMP) BMP Any of 1-15, the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin, any of agrin, semaphorin / brain degeneration proteins, spindle protein-1 and spindle protein-2, hepatocyte growth factor (HGF), ephrins, noggins, sonic hedgehogs, and any of the family of tyrosine hydroxylases.

[0115] Other useful transgenic products include proteins that regulate the immune system, including but not limited to cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 to IL-36 (including, for example, human interleukins IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12, IL-11, IL-12, IL-13, IL-18, IL-31, IL-35), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β and γ, stem cell factor, flk-2 / flt3 ligand. Gene products produced by the immune system can also be used in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, MHC class I and II molecules, and engineered immunoglobulins and MHC molecules. For example, in certain embodiments, rAAV antibodies can be designed to deliver canine or feline antibodies, such as anti-IgE, anti-IL31, anti-CD20, anti-NGF, anti-GnRH. Useful gene products also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor proteins (MCPs), decay accelerating factors (DAFs), CR1, CF2, CD59, and C1 esterase inhibitors (C1-INH).

[0116] Still other useful gene products include any one of the receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. The present invention encompasses receptors for cholesterol regulation and / or lipid regulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors, and scavenger receptors. The present invention also encompasses gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. Additionally, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box binding protein, interferon regulatory factor (IRF-1), Wilms' tumor protein, ETS binding proteins, STATs, GATA box binding proteins (e.g., GATA-3) and the forkhead family of winged helix proteins.

[0117] Other useful gene products include carbamyl synthetase I, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase (ASL) for the treatment of argininosuccinate lyase deficiency, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, rhesus alpha-fetoprotein (AFP), rhesus chorionic gonadotropin (CG), glucose-6-phosphatase, porphobilinogen deaminase, cystathionine beta synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin gene products [e.g., mini- or micro-dystrophin]. Still other useful gene products also include enzymes such as those that can be used in enzyme replacement therapy, which can be used for a variety of conditions caused by insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate can be used in the treatment of lysosomal storage diseases (e.g., suitable genes include genes encoding β-glucuronidase (GUSB)).

[0118] In certain embodiments, rAAV can be used in gene editing systems that can involve co-administration of one rAAV or multiple rAAV stocks. For example, rAAV can be engineered to deliver SpCas9, SaCas9, ARCUS, Cpf1, and other suitable gene editing constructs.

[0119] Still other useful gene products include gene products for treating hemophilia, including hemophilia B (comprising factor IX) and hemophilia A (comprising factor VIII and variants thereof, such as heterodimers and light and heavy chains with B domain deletions; U.S. Patent No. 6,200,560 and U.S. Patent No. 6,221,349). In some embodiments, the minigene includes the first 57 base pairs of the factor VIII heavy chain, which encodes a 10 amino acid signal sequence and a human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the minigene further includes the A1 and A2 domains and 5 amino acids from the N-terminus of the B domain and / or 85 amino acids from the C-terminus of the B domain and the A3, C1, and C2 domains. In yet other embodiments, nucleic acids encoding the Factor VIII heavy and light chains are provided in a single minigene separated by 42 nucleic acids encoding the 14 amino acids of the B domain [U.S. Patent No. 6,200,560].

[0120] Other useful gene products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having non-naturally occurring amino acid sequences containing insertions, deletions, or amino acid substitutions. For example, single-chain engineered immunoglobulins may be useful in certain immunocompromised patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce overexpression of a target.

[0121] Reducing and / or regulating gene expression is particularly desirable for the treatment of the hyperproliferative condition of disease (such as cancer and psoriasis) characterized by cell hyperproliferation. Target polypeptide comprises those polypeptides produced specifically in hyperproliferative cells or produced at a higher level compared with normal cells. Target antigen comprises polypeptides encoded by oncogenes such as myb, myc, fyn and translocation genes bcr / abl, ras, src, p53, neu, trk and EGRF. Except as the oncogene product of the target antigen, the target polypeptide for anticancer treatment and protection scheme comprises the variable region of the antibody produced by B cell lymphoma and the variable region of the T cell receptor of T cell lymphoma, and in some embodiments, the variable region is also used as the target antigen of autoimmune disease. Other tumor-associated polypeptides can also be used as target polypeptides, such as the polypeptides existing at a higher level in tumor cells, comprising polypeptides and folic acid binding polypeptides identified by monoclonal antibody 17-1A.

[0122] Other suitable therapeutic polypeptides and proteins include those that can be used to treat individuals with autoimmune diseases and disorders by conferring a broad-based protective immune response against targets associated with autoimmunity, including cell receptors and cells that produce "self" directed antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome ( syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, Wegner's granulomatosis, Crohn's disease, and ulcerative colitis. Each of these diseases is characterized by a T cell receptor (TCR) that binds to endogenous antigens and initiates the inflammatory cascade associated with autoimmune diseases.

[0123] Additional illustrative genes that can be delivered by rAAV include, but are not limited to, glucose-6-phosphatase, which is associated with glycogen storage disease or deficiency type 1A (GSD1); phosphoenolpyruvate carboxykinase (PEPCK), which is associated with PEPCK deficiency; cyclin-dependent kinase-like 5 (CDKL5), which is also known as serine / threonine kinase 9 (STK9), which is associated with epileptic seizures and severe neurodevelopmental disorders; galactose-1 phosphate uridylinositolase, which is associated with galactosemia; phenylalanine hydroxylase, which is associated with phenylketonuria (PKU); and Branched-chain alpha-ketoacid dehydrogenase associated with maple syrup urine disease; fumarylacetoacetate hydrolase associated with tyrosinemia type 1; methylmalonyl-CoA mutase associated with methylmalonic acidemia; medium-chain acyl-CoA dehydrogenase associated with medium-chain acetyl-CoA deficiency; ornithine transcarbamylase (OTC) associated with ornithine transcarbamylase deficiency; argininosuccinate synthetase (ASS1) associated with citrullinemia; lecithin cholesterol acyltransferase (LCAT) deficiency; methylmalonic acidemia (MMA); Niemann-Pick disease disease, type C1; propionic acidemia (PA); low-density lipoprotein receptor (LDLR) protein associated with familial hypercholesterolemia (FH); UDP-glucuronosyltransferase associated with Crigler-Najjar disease; adenosine deaminase associated with severe combined immunodeficiency; hypoxanthine guanine phosphoribosyltransferase associated with gout and Lesch-Nyhan syndrome; biotinidase associated with biotinidase deficiency; α-galactosidase A (α-GalA) associated with Fabry disease; ATP7B associated with Wilson's disease; β-glucocerebrosidase associated with Gaucher disease types 2 and 3; and Zellweger syndrome. syndrome); arylsulfatase A (ARSA) associated with degenerative leukodystrophy; galactocerebrosidase (GALC) 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 citrullinemia type II (CTLN2); carbamoyl phosphate synthase 1 (CPS1) associated with urea cycle disorders; survival motor neuron (SMN) protein associated with spinal muscular atrophy; and ceramidase associated with Farber lipogranulomatosis.beta-hexosaminidase associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff diseases; aspartylglucosaminidase associated with aspartylglucosidase; alpha-fucosidase associated with fucosidosis; alpha-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, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitors for the treatment of blocked blood vessels as seen, for example, in atherosclerosis, thrombosis, or embolism; and cytokinin for the treatment of Parkinson's disease. disease); beta-adrenergic receptors that are antisense to phospholamban, sarcoplasmic (endoplasmic) reticulum adenosine triphosphatase 2 (SERCA2), or mutant forms thereof; cardiac adenylate cyclase for the treatment of congestive heart failure; tumor suppressor genes, such as p53, for the treatment of various cancers; cytokines, such as various interleukins, for the treatment of inflammatory and immune disorders and cancer; dystrophin or mini-dystrophin and dystrophin-related protein or mini-dystrophin for the treatment of muscular dystrophy; and insulin or GLP-1 for the treatment of diabetes.

[0124] Additional genes and diseases of interest include, for example, dystonin gene-associated diseases such as hereditary sensory and autonomic neuropathy type VI (DST). The gene encodes dystonin; due to the size of the protein (approximately 7570 aa), dual AAV vectors may be required; SCN9A-associated diseases, in which loss of function mutants result in the inability to feel pain, and gain of function mutants cause painful conditions such as erythromelalgia. Another condition is Charcot-Marie-Tooth disease types 1F and 2E, which are characterized by progressive peripheral motor and sensory neuropathy with variable clinical and electrophysiological expression, due to mutations in the NEFL gene (neurofilament light chain).

[0125] In certain embodiments, the rAAV described herein can be used to treat mucopolysaccharidosis (MPS) disorders. Such rAAV can contain nucleic acid sequences encoding α-L-iduronidase (IDUA) for the treatment of MPS I (Hurler, Hurler-Scheie and Scheie syndromes); nucleic acid sequences encoding iduronate-2-sulfatase (IDS) for the treatment of MPS II (Hunter syndrome); nucleic acid sequences encoding sulfamidase (SGSH) for the treatment of MPS IIIA, B, C and D (Sanfilippo syndrome); nucleic acid sequences encoding N-acetylgalactosamine-6-sulfate sulfatase (GALNS) for the treatment of MPS IIIA and B (Morquio syndrome); nucleic acid sequences encoding N-acetylgalactosamine-6-sulfate sulfatase (GALNS) for the treatment of MPS VI (Maroteaux-Lamy syndrome). syndrome); a nucleic acid sequence encoding a hyaluronidase for the treatment of MPS IX (hyaluronidase deficiency); and a nucleic acid sequence encoding a β-glucuronidase for the treatment of MPS VII (Sly syndrome).

[0126] Immunogenic transgene

[0127] In some embodiments, rAAV vectors comprising nucleic acids encoding gene products associated with cancer (e.g., tumor suppressors) can be used to treat cancer by administering rAAV containing rAAV vectors to subjects with cancer. In some embodiments, rAAV vectors comprising nucleic acids encoding small interfering nucleic acids (e.g., shRNA, miRNA) that inhibit the expression of gene products associated with cancer (e.g., oncogenes) can be used to treat cancer by administering rAAV containing rAAV vectors to subjects with cancer. In some embodiments, rAAV vectors comprising nucleic acids encoding gene products associated with cancer (or functional RNAs that inhibit the expression of genes associated with cancer) can be used for research purposes, such as studying cancer or identifying therapeutic agents for treating cancer. The following is a non-limiting list of exemplary genes (e.g., oncogenes and tumor suppressors) known to be associated with the development of cancer: AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, AT P5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B , CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C, COX7A2, CRAT, CRHR1, C SF1R, 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,<h2 style=";text-align:left;direction:ltr">F2R、FASTK、FBN1、FBN2、FES、FGFR1、FGR、FKBP8、FN1、FOS、FOSL1、FOSL2、FO XG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GD F15、GNA13、GNAS、GNB2、GNB2L1、GPR39、GRB2、GSK3A、GSPT1、GTF2I、HDAC1、 HDGF、HMMR、HPRT1、HRB、HSPA4、HSPA5、HSPA8、HSPB1、HSPH1、HYAL1、HYOU1、I CAM1, ID1, ID2, IDUA, IER3, IFITM1, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3, ITGA3, ITGA6, ITGB4, JAK1, JARID1A, JUN, JUNB, JU ND、K-α-1、KIT、KITLG、KLK10、KPNA2、KRAS2、KRT18、KRT2A、KRT9、LAMB1、LA MP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF、LYN、LZTR1、MADH1、MAP2K2、MAP3K8 、MAPK12、MAPK13、MAPKAPK3、MAPRE1、MARS、MAS1、MCC、MCM2、MCM4、MDM2、MD M4、MET、MGST1、MICB、MLLT3、MME、MMP1、MMP14、MMP17、MMP2、MNDA、MSH2、MS H6、MT3、MYB、MYBL1、MYBL2、MYC、MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、NF1 、NF2、NFKB1、NFKB2、NFSF7、NID、NINE、NMBR、NME1、NME2、NME3、NOTCH1、NOTC H2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、NSEP1、OSM、PA2G 4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PEA15、 PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、PIM1、PKM2、PKMYT1、PL K2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN, PTPRN, RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RAR B. RASGRF1, RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6 KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, Stimulus Protein Protein Dispersed Pain1(SMPD1), SNAI2, S.K ND1, SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STA T2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFD P2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, T LE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1 TOB1, TP53, TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101 TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1. WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9.

[0128] The rAAV vector may include a nucleic acid encoding a protein or functional RNA that regulates apoptosis as a transgene. The following is a non-limiting list of genes associated with apoptosis, and nucleic acids encoding the products of these genes and their homologs, as well as small interfering nucleic acids (e.g., shRNA, miRNA) that inhibit the expression of these genes and their homologs, are used as transgenes in certain embodiments of the present invention: RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNI P3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CAS P4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A, GDNF, HRK, I GF1R, LTA, LTBR, ​​MCL1, NOL3, PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRS F12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFS F14, TNFSF18, CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4 and TRAF5.

[0129] Useful transgenic products also include miRNA. MiRNA and other small interfering nucleic acids regulate gene expression by target RNA transcript cracking / degradation or translation inhibition of target messenger RNA (mRNA). MiRNA is naturally expressed, usually as final 19-25 kinds of non-translated RNA products. MiRNA shows its activity by sequence-specific interactions with the 3' untranslated region (UTR) of the target mRNA. These endogenously expressed miRNAs form hairpin precursors, which are subsequently processed into miRNA duplexes and further processed into "mature" single-stranded miRNA molecules. This mature miRNA guides the multiprotein complex miRISC, which identifies the target site of the target mRNA based on complementarity with the mature miRNA, for example, in the 3' UTR region.

[0130] In certain embodiments of the methods, the following non-limiting list of miRNA genes and their homologs can be used as targets for transgenes or small interfering nucleic acids (e.g., miRNA sponges, antisense oligonucleotides, TuD RNA) encoded by transgenes: 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-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-106a, hsa-miR-106a * , hsa-miR-106b, hsa-miR-106b * , hsa-miR-107, hsa-miR-10a, hsa-miR-10a * 、hsa-miR-10b、hsa-miR-10b *hsa-miR-1178, hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR -1201、hsa-miR-1202、hsa-miR-1203、hsa-miR-1204、hsa-miR-1205、hsa-miR-1206、hsa-miR-1207-3p、hsa-miR-1207-5p、hsa-miR-1208、hsa-miR-122、hsa-miR-122 * hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p, hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226 * hsa-miR-1227, hsa-miR-1228, hsa-miR-1228 * hsa-miR-1229, hsa-miR-1231, hsa-miR-1233, hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124, hsa-miR-124 * hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246, hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR-1253, hsa- miR-1254、hsa-miR-1255a、hsa-miR-1255b、hsa-miR-1256、hsa-miR-1257、hsa-miR-125 8、hsa-miR-1259、hsa-miR-125a-3p、hsa-miR-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-miR-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, hsa-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-1287, hsa-miR-1288, hsa-miR-1289, hsa-miR-129 * hsa-miR-1290, hsa-miR-1291, hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa -miR-1299, hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304, hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a, hsa-miR-130a * 、hsa-miR-130b、hsa-miR-130b * hsa-miR-132 hsa-miR-132 * hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, 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-181a * hsa-miR-181a-2* hsa-miR-181b, hsa-miR-181c, hsa-miR-181c * hsa-miR-181d, hsa-miR-182, hsa-miR-182 * hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183 * hsa-miR-184, hsa-miR-185, hsa-miR-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-192 hsa-miR-192 * 、hsa-miR-193a-3p、hsa-miR-193a-5p、hsa-miR-193b、hsa-miR-193b * hsa-miR-194 hsa-miR-194 * hsa-miR-195 hsa-miR-195 * 、hsa-miR-196a、hsa-miR-196a * hsa-miR-196b, hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a * hsa-miR-19b hsa-miR-19b-1 * hsa-miR-19b-2 * hsa-miR-200a hsa-miR-200a * hsa-miR-200b hsa-miR-200b * 、hsa-miR-200c、hsa-miR-200c* hsa-miR-202 hsa-miR-202 * hsa-miR-203, hsa-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 * hsa-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-219-2-3p、hsa-miR-219-5p、hsa-miR-22、hsa-miR-22 * hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221, hsa-miR-221 * hsa-miR-222 hsa-miR-222 * hsa-miR-223 hsa-miR-223 * 、hsa-miR-224、hsa-miR-23a、hsa-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-miR-27b hsa-miR-27b * hsa-miR-28-3p, hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298, hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a * hsa-miR-29b, hsa-miR-296-1 * hsa-miR-296-2 * hsa-miR-29c hsa-miR-29c * 、hsa-miR-300、hsa-miR-301a、hsa-miR-301b、hsa-miR-302a、hsa-miR-302a * 、hsa-miR-302b、hsa-miR-302b * 、hsa-miR-302c、hsa-miR-302c * hsa-miR-302d hsa-miR-302d * 、hsa-miR-302e、hsa-miR-302f、hsa-miR-30a、hsa-miR-30a * hsa-miR-30b hsa-miR-30b * hsa-miR-30c, hsa-miR-30c-1 * hsa-miR-30c-2 * hsa-miR-30d 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, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p, hsa-miR-362-5p, hsa-miR-363, hsa-miR-363 * hsa-miR-365, hsa-miR-367, hsa-miR-367 * hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373 * 、hsa-miR-374a、hsa-miR-374a * 、hsa-miR-374b、hsa-miR-374b * hsa-miR-375, hsa-miR-376a, hsa-miR-376a* hsa-miR-376b, hsa-miR-376c, hsa-miR-377, hsa-miR-377 * hsa-miR-378 hsa-miR-378 * hsa-miR-379 hsa-miR-379 * hsa-miR-380 hsa-miR-380 * hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411, hsa-miR-411 * hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424, hsa-miR-424 * hsa-miR-425 hsa-miR-425 * 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-452 * 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-490-3p, 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-miR-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-508-5p, hsa-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, hsa-miR-513b, hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p, hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517 * 、hsa-miR-517a、hsa-miR-517b、hsa-miR-517c、hsa-miR-518a-3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c * 、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-miR-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-3p、hsa-miR-5486-5p、hsa-miR-548c-3p 、hsa-miR-548c-5p、hsa-miR-548d-3p、hsa-miR-548d-5p、hsa-miR-548e、hsa-miR-548f、hsa-m iR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j, hsa-miR-548k, hsa-miR-5481, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550 * 、hsa-miR-551a、hsa-miR-551b、hsa-miR-551b *hsa-miR-552, hsa-miR-553, hsa-miR-554, hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558, hsa-miR-559, hsa-miR-561, hs a-miR-562, hsa-miR-563, hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570, hsa-miR-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-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606, hs a-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-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624, hsa-miR-624 * hsa-miR-625 hsa-miR-625 * hsa-miR-626, hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629 * hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hs a-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 * hsa-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-874, 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-885-5p, hsa-miR-886-3p, hsa-miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888 * , hsa-miR-889, hsa-miR-890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9, hsa-miR-9 * , hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-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-miR-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, of interest may be miRNAs that target chromosome 8 open reading frame 72 (C9orf72), which expresses superoxide dismutase (SOD1), which is associated with amyotrophic lateral sclerosis (ALS).

[0131] MiRNA suppresses the function of the mRNA of its targeting, and therefore suppresses the expression of the polypeptide encoded by mRNA.Therefore, (partially or entirely) blocking the activity of miRNA (for example, silencing miRNA) can effectively induce or restore the expression of the suppressed polypeptide (making the polypeptide go to suppress). In one embodiment, the miRNA activity in any of the inhibitory cells in a variety of methods is realized to suppress the polypeptide encoded by the mRNA target of miRNA. For example, the activity of blocking miRNA can be realized by hybridizing with the small interfering nucleic acids (for example, antisense oligonucleotides, miRNA sponges, TuD RNA) that are complementary or substantially complementary to miRNA, thus blocking the interaction of miRNA and its target mRNA. As used herein, the small interfering nucleic acids that are substantially complementary to miRNA are small interfering nucleic acids that can hybridize with miRNA and block the activity of miRNA. In some embodiments, a small interfering nucleic acid that is substantially complementary to a miRNA is a small interfering nucleic acid that is completely complementary to the miRNA except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 bases. "MiRNA inhibitors" are agents that block miRNA function, expression and / or processing. For example, these molecules include, but are not limited to, microRNA-specific antisense molecules, microRNA sponges, strong decoy RNA (TuD RNA) and microRNA oligonucleotides (double-stranded, hairpins, short oligonucleotides) that inhibit the interaction of miRNA with the Drosha complex.

[0132] Still other useful transgenes can include transgenes that encode immunoglobulins that confer passive immunity to pathogens." Immunoglobulin molecules " are proteins containing the immunologically active portion of heavy and light immunoglobulin chains that are covalently coupled together and can be combined with antigen specificity. Immunoglobulin molecules can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), classification (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The terms "antibody" and "immunoglobulin" can be used interchangeably in this article.

[0133] An "immunoglobulin heavy chain" is a polypeptide comprising at least a portion of an immunoglobulin antigen-binding domain and at least a portion of an immunoglobulin heavy chain variable region or at least a portion of an immunoglobulin heavy chain constant region. Therefore, immunoglobulin-derived heavy chains share significant regions of amino acid sequence homology with members of the immunoglobulin gene superfamily. For example, the heavy chain in a Fab fragment is an immunoglobulin-derived heavy chain.

[0134] An "immunoglobulin light chain" is a polypeptide that contains at least a portion of an immunoglobulin antigen-binding domain and at least a portion of an immunoglobulin light chain variable region or at least a portion of a constant region. Therefore, immunoglobulin-derived light chains share significant regions of amino acid homology with members of the immunoglobulin gene superfamily.

[0135] "Immunoadhesins" are chimeric antibody-like molecules that combine the functional domains of a binding protein (usually a receptor, ligand, or cell adhesion molecule) with immunoglobulin constant domains, typically comprising a hinge and Fc region.

[0136] A "fragment antigen-binding (Fab) fragment" is a region of an antibody that binds to an antigen. It is composed of one constant domain and one variable domain of each of the heavy and light chains.

[0137] The antipathogen constructs are selected based on the causative agent / pathogen of the disease against which protection is sought. These pathogens can be of viral, bacterial or fungal origin and can be used to prevent humans from contracting human diseases or to prevent veterinary diseases in non-human mammals or other animals.

[0138] rAAV can contain genes encoding antibodies and, in particular, neutralizing antibodies against viral pathogens. Such antiviral antibodies can include anti-influenza antibodies against one or more of influenza A, influenza B, and influenza C. Type A viruses are the most virulent human pathogens. Serotypes of influenza A associated with epidemics include: H1N1, which caused the Spanish flu in 1918 and the swine flu in 2009; H2N2, which caused the Asian flu in 1957; H3N2, which caused the Hong Kong flu in 1968; H5N1, which caused the avian flu in 2004; H7N7; H1N2; H9N2; H7N2; H7N3; and H10N7. Other target pathogenic viruses include: arenaviruses (including funin, machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picornoviridae (including rhinoviruses, echoviruses), coronaviruses, paramyxoviruses, measles virus, respiratory syncytial virus, enveloped viruses, coxsackieviruses, JC virus, parvovirus B19, paramyxoviruses, and rubella virus. Influenza virus, adenovirus, reovirus, variola virus (smallpox) and cowpox virus (Vaccinia / Cowpox) from the poxvirus family, and varicella-zoster virus (pseudorabies). Viral hemorrhagic fevers are caused by members of the arenavirus family (Lassa fever) (this family is also related to lymphocytic choriomeningitis virus (LCM)), filoviruses (Ebola virus), and hantaviruses (puremala virus). Members of the picornaviruses (a subfamily of rhinoviruses) are associated with the common cold in humans. The coronavirus family includes a variety of non-human viruses, such as infectious bronchitis virus (poultry), transmissible gastroenteritis virus (pigs), porcine hemagglutinin encephalomyelitis virus (pigs), feline infectious peritonitis virus (cats), feline enteric coronavirus (cats), and canine coronavirus (dogs). Human respiratory coronaviruses have been postulated to be associated with the common cold, hepatitis non-A, B, or C, and severe acute respiratory syndrome (SARS). Paramyxovirus family Includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, mumps virus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, measles virus (including measles and canine distemper) and pneumovirus (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).Thus, in certain embodiments, the rAAV vectors described herein can be engineered to express anti-Ebola antibodies (e.g., 2G4, 4G7, 13C6), anti-influenza antibodies (e.g., FI6, CR8033), and anti-RSV antibodies (e.g., palivizumab, motavizumab). Neutralizing antibody constructs directed against bacterial pathogens can 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 a toxin produced by the bacteria.Examples of airborne bacterial pathogens include, e.g., Neisseria meningitidis (meningitis), Klebsiella pneumonia (pneumonia), Pseudomonas aeruginosa (pneumonia), Pseudomonas pseudomallei (pneumonia), Pseudomonas mallei (pneumonia), Acinetobacter (pneumonia), Moraxella catarrhalis, Moraxella lacunata, Alkaligenes, Cardiobacterium, Haemophilus influenzae (influenza), Haemophilus parainfluenzae, Bordetella pertussis (whooping cough), Francisella tularensis (whooping cough), and Pseudomonas aeruginosa (pneumonia). tularensis (pneumonia / fever), Legionella pneumonia (Legionnellipneumonia), Chlamydia psittaci (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis (Tuberculosis (TB)), Mycobacterium kansasii (TB), Mycobacterium avium (pneumonia), Nocardia asteroides (pneumonia), Bacillus anthracis (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacteria diphtheria (diphtheria), Mycoplasma pneumoniae (pneumonia).

[0139] rAAV can contain genes encoding antibodies and specifically neutralizing antibodies against bacterial pathogens such as the causative agent of anthrax, i.e., the toxin produced by Bacillus anthracis. Neutralizing antibodies have been described against protectant (PA), one of the three peptides that form the toxoid. The other two polypeptides consist of lethal factor (LF) and edema factor (EF). Anti-PA neutralizing antibodies have been described as effective for passive immunization against anthrax. See, e.g., U.S. Patent No. 7,442,373; R. Sawada-Hirai et al., J Immune Based Ther Vaccines 2004; 2:5. (online May 12, 2004). Still other anti-anthrax toxin neutralizing antibodies have been described and / or can be produced. Similarly, neutralizing antibodies against other bacteria and / or bacterial toxins can be used to produce AAV-deliverable anti-pathogen constructs as described herein.

[0140] Antibodies against infectious diseases can be caused by parasites or fungi, including, for example, Aspergillus species, Absidia corymbifera, Rhixpus stolonifer, Mucorplumbeaus, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Penicillium species, Micropolyspora faeni, Thermoactinomyces vulgaris, Alternaria alternate, Cladosporium species, Helminthosporium, and Stachybotrys species.

[0141] rAAV can contain genes encoding antibodies and neutralizing antibodies specifically against the causative agents of the following 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 can be, for example, but are not limited to, α-synuclein, anti-vascular endothelial growth factor (VEGF) (anti-VEGF), anti-VEGFA, anti-PD-1, anti-PDL1, anti-CTLA-4, anti-TNF-α, anti-IL-17, anti-IL-23, anti-IL-21, anti-IL-6, anti-IL-6 receptor, anti-IL-5, anti-IL-7, anti-factor XII, anti-IL-2, anti-HIV, anti-IgE, anti-tumor necrosis factor receptor 1 (TNFR1), anti-notch 2 / 3, anti-notch 1, anti-OX40, anti-erb-b2 receptor tyrosine kinase 3 (ErbB3), anti-ErbB2, anti-β cell maturation antigen, anti-B lymphocyte stimulator, anti-CD20, anti-HER2, anti-granulocyte macrophage colony stimulating factor, anti-oncostatin M (OSM), anti-lymphocyte activation gene 3 (LAG3) protein, anti-CCL20, anti-serum amyloid protein P component (SAP), anti-prolyl hydroxylase inhibitors, anti-CD38, anti-glycoprotein IIb / IIIa, anti-CD52, anti-CD30, anti-IL-1β, anti-epidermal growth factor receptor, anti-CD25, anti-RANK ligand, anti-complement system protein C5, anti-CD11a, anti-CD3 receptor, anti-alpha-4 (α4) integrin, anti-RSV F protein and anti-integrin α4β7. Still other pathogens and diseases will be apparent to those skilled in the art. Other suitable antibodies can include antibodies useful for treating Alzheimer's disease, such as anti-β amyloid (e.g., crenezumab, solanezumab, aducanumab), anti-β amyloid fibrils, anti-β amyloid plaques, anti-tau, bapineuzamab, and other antibodies. Other suitable antibodies for treating various indications include those described, for example, in PCT / US2016 / 058968, filed October 27, 2016, disclosed as WO 2017 / 075119A1.

[0142] II. rAAV vector production

[0143] For the production of AAV viral vectors (e.g., recombinant (r) AAV), the expression cassette can be carried on any suitable vector (e.g., plasmid) for delivery to a packaging host cell. The plasmids that can be used in the present invention can be engineered so that they are suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, and other cells. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art.

[0144] Methods for producing and isolating AAV suitable for use as a vector are known in the art. See generally, for example, Grieger and Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications", Adv. Biochem. Engin / Biotechnol 99: 119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med 10: 717-733; and references cited below, each of which is incorporated herein by reference in its entirety. In order to package the transgene into virions, ITR is the only AAV component in cis required in the construct identical to the nucleic acid molecule containing the expression cassette. The cap and rep genes can be supplied in trans.

[0145] In one embodiment, expression cassette described herein is engineered to genetic element (for example, shuttle plasmid), and the transgenic construct sequence carried on it is transferred to packaging host cell to produce viral vector. In one embodiment, selected genetic element can be delivered to AAV packaging cell by any suitable method, and the method comprises transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA coated pellet, viral infection and protoplast fusion. Stable AAV packaging cell can also be prepared. Alternatively, expression cassette can be used to produce viral vector except AAV. The method for preparing such construct is known to nucleic acid manipulation technicians and comprises genetic engineering, recombinant engineering and synthetic technology. See, for example, " Molecular Cloning: A Laboratory Manual ", edited by Green and Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012).

[0146] The term "AAV intermediate" or "AAV vector intermediate" refers to assembled rAAV capsids that lack the desired genomic sequence packaged therein. These may also be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette, or contain only partially packaged genomic sequence that is insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to the host cell.

[0147] Recombinant adeno-associated viruses (AAVs) described herein can be produced using known techniques. See, for example, WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; US 7588772 B2. Such methods involve culturing host cells containing nucleic acid sequences encoding AAV capsid proteins; functional rep genes; expression cassettes consisting of at least AAV inverted terminal repeats (ITRs) and transgenes; and auxiliary functions sufficient to allow the expression cassettes to be packaged into AAV capsid proteins. Methods for producing capsids, their coding sequences, and methods for producing rAAV viral vectors have been described. See, for example, Gao et al., Proceedings of the National Academy of Sciences of the United States of America 100(10):6081-6086(2003) and US2013 / 0045186A1.

[0148] In one embodiment, a production cell culture that can be used to produce recombinant AAV is provided. This cell culture contains nucleic acids that express AAV capsid proteins in host cells; nucleic acid molecules suitable for packaging into AAV capsids, such as vector genomes containing AAV ITR and non-AAV nucleic acid sequences encoding gene products, wherein the non-AAV nucleic acid sequences are operably linked to sequences for guiding product expression in host cells; and AAV rep functions and adenovirus helper functions sufficient to allow nucleic acid molecules to be packaged into recombinant AAV capsids. In one embodiment, the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells and other cells) or insect cells (e.g., baculovirus).

[0149] Optionally, the rep function is provided by an AAV other than the AAV that provides the capsid. For example, rep can be, but is not limited to, an AAV1 rep protein, an AAV2 rep protein, an AAV3 rep protein, an AAV4 rep protein, an AAV5 rep protein, an AAV6 rep protein, an AAV7 rep protein, an AAV8 rep protein; or rep 78, rep 68, rep 52, rep 40, rep68 / 78, and rep40 / 52; or a fragment thereof; or another source. Optionally, the rep and cap sequences are located on the same genetic element in cell culture. A spacer may be present between the rep sequence and the cap gene. Any of these AAV or mutant AAV capsid sequences can be under the control of exogenous regulatory control sequences that direct their expression in the host cell.

[0150] In one embodiment, cells are manufactured in suitable cell culture (e.g., HEK 293) cells. The method for manufacturing the gene therapy vector described herein includes methods well known in the art, such as producing plasmid DNA, producing vectors, and purifying vectors for producing gene therapy vectors. In certain embodiments, the gene therapy vector is an AAV vector, and the plasmid produced is an AAV cis plasmid encoding the vector genome comprising the gene of interest, an AAV trans plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may include method steps, such as starting cell culture, performing cell passage, inoculating cells, transfecting cells with plasmid DNA, exchanging the post-transfection culture medium for serum-free culture medium, and collecting cells and culture medium containing the vector. The collected cells and culture medium containing the vector are referred to as crude cell collections in this article. In another system, the gene therapy vector is introduced into insect cells by being infected with a baculovirus-based vector. For a review of these production systems, see generally, for example, Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each 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.

[0151] Thereafter, the crude cell collection can be subjected to the subject method steps of, for example, concentrating the vector collection, diafiltering the vector collection, microfluidizing the vector collection, nuclease digesting the vector collection, filtering the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare large quantities of vector.

[0152] Two-step affinity chromatography purification at high salt concentrations is followed by anion exchange resin chromatography to purify the vector drug product and remove empty capsids. These methods are described in more detail in International Patent Publication No. WO 2017 / 160360, which is incorporated herein by reference. For AAV8, purification methods are described in Publication No. WO 2017 / 100676, for rh10, in International Patent Publication No. WO 2017 / 100704, and for AAV1, in International Patent Publication No. WO 2017 / 100674, which are incorporated herein by reference in their entireties.

[0153] To calculate the content of empty and intact particles, the VP3 band volume of the selected sample (e.g., in the examples herein, the preparation purified by iodixanol gradient, where GC# = particle #) 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 article peak. The number of particles per 20 μL loaded (pt) is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL - GC / mL gives empty pt / mL. Empty pt / mL is divided by pt / mL and ×100 to obtain the percentage of empty particles.

[0154] In general, methods for determining empty capsids and AAV vector particles with packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6: 1322-1330; Sommer et al., Molec. Ther. (2003) 7: 122-128. To test denatured capsids, the method comprises subjecting the treated AAV stock solution to SDS-polyacrylamide gel electrophoresis (consisting of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% triacetate in a buffer), then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins, preferably anti-AAV capsid monoclonal antibodies, most preferably B1 anti-AAV2 monoclonal antibodies (Wobus et al., Journal of Virology (2000) 74: 9281-9293). Then use secondary antibody, described secondary antibody is combined with primary antibody and contains a kind of device for detecting the combination with primary antibody, more preferably the anti-IgG antibody containing the detection molecule covalently bound thereto, most preferably the sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase.A kind of method for detecting combination is used for semi-quantitatively determining the combination between primary antibody and secondary antibody, preferably the detection method that can detect radioisotope emission, electromagnetic radiation or colorimetric change, most preferably chemiluminescence detection kit.For example, for SDS-PAGE, sample can be extracted from column fraction and heated in SDS-PAGE loading buffer containing reducing agent (for example, DTT), and capsid protein is resolved on prefabricated gradient polyacrylamide gel (for example, Novex).Silver staining can be carried out using SilverXpress (Invitrogen, California) or other suitable staining methods (i.e. SYPRO ruby ​​or Coomassie staining) according to the manufacturer's instructions.In one embodiment, the concentration of the AAV vector genome (vg) in the 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, primers and TaqMan PCR products specific for the DNA sequence between the primers are used. TMThe fluorescent probe is further diluted and amplified. The number of cycles (threshold cycles, Ct) required for each sample to reach a defined fluorescence level is measured on an Applied Biosystems Prism7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it relative to the Ct value of the plasmid standard curve. Endpoint determination based on digital PCR can also be used.

[0155] On the one hand, an optimized q-PCR method is used, which utilizes a broad spectrum serine protease, such as proteinase K (as commercially available from Qiagen). 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 proteinase K buffer and treated with proteinase K, and then heat inactivated. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. Proteinase K buffer can be concentrated 2 times or more. Typically, proteinase K is treated at about 0.2 mg / mL, but can vary between 0.1 g / mL and about 1 mg / mL. The treatment step is typically performed at about 55°C for about 15 minutes, but can be performed at lower temperatures (e.g., about 37°C to about 50°C) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60°C) for a shorter period of time (e.g., about 5 to 10 minutes). Similarly, heat inactivation is typically performed at about 95°C for about 15 minutes, but the temperature can be lowered (e.g., from about 70°C to about 90°C) and the time can be extended (e.g., from about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

[0156] Additionally or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining the titer of single-stranded and self-complementary AAV vector genomes by ddPCR have been described. See, for example, M. Lock et al., Human Gene Therapy Methods. 2014 Apr; 25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0157] Briefly, a method for separating rAAV particles having packaged genomic sequences from genome-defective AAV intermediates involves subjecting a suspension comprising recombinant AAV viral particles and AAV capsid protein intermediates to high performance liquid chromatography, wherein the AAV viral 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 UV absorbance of the eluate at about 260 and about 280. The pH can be adjusted based on the selected AAV. See, for example, WO2017 / 160360 (AAV9), WO2017 / 100704 (AAVrh10), WO 2017 / 100676 (e.g., AAV8), and WO2017 / 100674 (AAV1), which are incorporated herein by reference. In this method, AAV intact capsids are collected from the eluted fractions when the A260 / A280 ratio reaches an inflection point. In one example, for the affinity chromatography step, the diafiltered product can be applied to a Capture Select ELISA that efficiently captures AAV2 serotypes. TM Poros-AAV2 / 9 affinity resin (Life Technologies) was used. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins passed through the column, while AAV particles were efficiently captured.

[0158] III. Compositions and Uses

[0159] Provided herein is a composition comprising at least one rAAV stock solution (e.g., rAAV stock solution or mutant rAAV stock solution) and optional carriers, excipients and / or preservatives. The rAAV stock solution refers to a plurality of rAAV vectors, the amount of the plurality of rAAV vectors being the same as described, for example, in the discussion below regarding concentrations and dosage units.

[0160] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, vehicle solutions, suspensions, colloids, and the like. The use of such vehicles 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, vesicles, etc.) can be used to introduce the compositions of the present invention into suitable host cells. In particular, the transgene delivered by the rAAV vector can be formulated for delivery, wherein the transgene is encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0161] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, such as an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be shipped as a concentrate that is diluted for administration to a subject. In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0162] A suitable surfactant or combination of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated with a primary hydroxyl group is selected, such as F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers can be selected, namely nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylylglyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are typically named with the letter "P" (for poloxamer) followed by three numbers: the first two digits x 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0163] Vector is administered in sufficient amounts to transfect cells and provide sufficient levels of gene transfer and expression, so that therapeutic benefit is provided, without excessive side effects or with medically acceptable physiological effects, which can be determined by the technical staff of the medical field. Conventional and pharmaceutically acceptable route of administration include but are not limited to being directly delivered to desired organs (for example, liver (optionally by hepatic artery), lung, heart, eyes, kidney), oral, inhaled, intranasal, intrathecal, intratracheal, intraarterial, directly delivered to eyes (optionally by eye delivery, subretinal injection, intraretinal injection, in vitreous body, local), intravenous, intramuscular, subcutaneous, intradermal and other parent route of administration. In one embodiment, route of administration is subretinal or intravitreal injection. If desired, route of administration can be combined.

[0164] The dosage of the viral vector will depend primarily on factors such as the condition being treated, the patient's age, weight, and health, and therefore may vary between patients. For example, a therapeutically effective human dose of a viral vector will typically be in the range of about 25 to about 1000 microliters to about 100 mL of a solution containing a concentration of about 1×10 9 to 1×10 16 Genomic viral vectors. The dosage will be adjusted to balance the therapeutic benefit with any side effects, and this dosage can vary depending on the therapeutic application for which the recombinant vector is employed. The expression level of the transgene can be monitored to determine the dosage frequency of the resulting viral vector, preferably an AAV vector containing a minigene. Optionally, a dosage regimen similar to that described for therapeutic purposes can be used for immunization using the compositions of the present invention.

[0165] The replication-defective virus composition can be formulated in dosage units to contain about 1.0×10 9 GC to about 1.0×10 16 The range of GC is 1.0×10 GC (to treat subjects with an average weight of 70 kg), including all integer or fractional amounts within the range, and for human patients, preferably 1.0×10 12 GC to 1.0×10 14 GC. In one embodiment, the composition is formulated to contain at least 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 GC, including all integers and fractions within the range. In another embodiment, the composition is formulated to contain at least 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 or 9×10 10 GC, including all integers and fractions within the range. In another embodiment, the composition is formulated to contain at least 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11, 6×10 11 , 7×10 11 , 8×10 11 or 9×10 11 GC, including all integers and fractions within the range. In another embodiment, the composition is formulated to contain at least 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 or 9×10 12 GC, including all integers and fractions within the range. In another embodiment, the composition is formulated to contain at least 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 or 9×10 13 GC, including all integers and fractions within the range. In another embodiment, the composition is formulated to contain at least 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 or 9×10 14 GC, including all integers and fractions within the range. In another embodiment, the composition is formulated to contain at least 1×10 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 or 9×10 15 GC, including all integers and fractions within the stated range. In one embodiment, for human use, the dosage range may be 1×10 10 to about 1×10 12 GC, including all integer or fractional quantities in the stated range.

[0166] These aforementioned doses can be administered in various volumes of carrier, excipient, or buffer formulations, ranging from about 25 to about 1000 microliters or more, inclusive, depending on the size of the area to be treated, the titer of the virus used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 375 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 550 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 650 μL. In another embodiment, the volume is about 700 μL. In another embodiment, the volume is between about 700 and about 1000 μL.

[0167] In certain embodiments, the dose may be in the range of about 1×10 9 GC / g brain mass to about 1×10 12 GC / g brain mass. In certain embodiments, the dose may be in the range of about 3×10 10 GC / g brain mass to about 3×10 11 GC / g brain mass. In certain embodiments, the dose may be in the range of about 5×10 10 GC / g brain mass to about 1.85×10 11 GC / g brain mass.

[0168] In another aspect, an aqueous suspension suitable for administration to a subject is provided. In one embodiment, the suspension comprises an aqueous suspending fluid and about 1×10 9 virus particles to about 1×10 13GC or viral particles per eye of a recombinant adeno-associated virus (rAAV) as described herein, which can be used as a therapeutic agent for treating or preventing ocular diseases. In one embodiment, the suspension is suitable for subretinal or intravitreal injection.

[0169] In one embodiment, the viral construct can be expressed at a rate of at least about 1×10 9 GC to about 1×10 15 GC or about 1×10 11 GC to 5×10 13 Dose delivery of GC. Suitable volumes for delivering these doses and concentrations can be determined by those skilled in the art. For example, a volume of about 1 μL to 150 mL can be selected, with larger volumes being selected for adults. Typically, a suitable volume is about 0.5 mL to about 10 mL for newborn infants, and about 0.5 mL to about 15 mL for older infants. For young children, a volume of about 0.5 mL to about 20 mL can be selected. For children, a volume of up to about 30 mL can be selected. For prepubertal teenagers and adolescents, a volume of up to about 50 mL can be selected. In still other embodiments, the volume selected for intrathecal administration of the patient can be about 5 mL to about 15 mL or about 7.5 mL to about 10 mL. Other suitable volumes and dosages can be determined. The dosage will be adjusted to balance the therapeutic benefits with any side effects, and this dosage can vary depending on the therapeutic application employing the recombinant vector.

[0170] The recombinant vector described above can be delivered to a host cell according to the disclosed methods. The rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or salt mixture. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH within this range may be desired for intrathecal delivery; and for intravenous, subretinal, or intravitreal injection, a pH of about 6.8 to about 7.2 may be desired. However, other pHs within the widest range and these subranges may be selected for other delivery routes.

[0171] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an adjuvant. In view of the indication for which the virus is to be transferred, a person skilled in the art can easily select a suitable carrier. For example, a suitable carrier comprises saline, which can be formulated with a variety of 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 include components that prevent rAAV from adhering to the infusion tube but do not interfere with the binding activity of rAAV in vivo. Suitable surfactants or combinations of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminating in a primary hydroxyl group is selected, such as F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers can be selected, namely nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylylglyceride), polyoxyoleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are typically named with the letter "P" (for poloxamer) followed by three numbers: the first two digits x 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 was selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension. In one example, the formulation may contain, for example, a buffered saline solution comprising one or more of sodium chloride, sodium bicarbonate, dextran, magnesium sulfate (e.g., magnesium sulfate·7H2O), potassium chloride, calcium chloride (e.g., calcium chloride·2H2O), disodium hydrogen phosphate, and mixtures thereof in water. Suitably, for intrathecal delivery, the osmolarity is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as a suspending agent, or in combination with another suspending agent and other optional excipients. See, e.g., Elliotts Solution [Lukare Medical]. In other embodiments, the formulation may contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0172] Optionally, the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and one or more carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, methylparaben, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0173] The compositions according to the present invention may include a pharmaceutically acceptable carrier, as defined above. Suitably, the compositions described herein include an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed to be delivered to a subject by injection, an osmotic pump, an intrathecal catheter, or by another device or route of delivery. In one example, the composition is formulated for intrathecal delivery.

[0174] As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of drug administration by injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery can include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital / intracisternal and / or C1-2 puncture. For example, the material can be introduced via a lumbar puncture to diffuse throughout the subarachnoid space. In another example, the material can be injected into the cisterna magna.

[0175] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the administration of a drug directly into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically by suboccipital puncture or by injection directly into the cisterna magna or by a permanently placed tube.

[0176] On the one hand, the carrier provided herein can be administered intrathecally by the method and / or device.See, for example, WO2017 / 181113, which is incorporated herein by reference. Alternatively, other devices and methods can be selected. The method comprises the following steps: a spinal needle is advanced into the patient's cisterna magna; a flexible tube of a certain length is connected to the proximal hub of the spinal needle, and the output port of the valve is connected to the proximal end of the flexible tube, and after carrying out the advancement and connection steps, and after allowing the tube to use the patient's cerebrospinal fluid for self-priming, a first container containing a certain amount of isotonic solution is connected to the flushing inlet of the valve, and then a second container containing a certain amount of pharmaceutical composition is connected to the carrier inlet of the valve. After the first blood vessel and the second blood vessel are connected to the valve, a fluid flow path is opened between the carrier inlet and outlet of the valve, and the pharmaceutical composition is injected into the patient by the spinal needle, and after injecting the pharmaceutical composition, a fluid flow path is opened by the flushing inlet and outlet of the valve, and an isotonic solution is injected into the spinal needle to flush the pharmaceutical composition into the patient.

[0177] This method and this device can each optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices can be used for such intrathecal delivery.

[0178] It should be noted that the term "a" or "an" refers to one or more. Thus, the terms "a or an," "one or more," and "at least one" are used interchangeably herein.

[0179] The words "comprise," "comprises," and "comprising" are to be interpreted as inclusive rather than exclusive. The words "consist of," "consisting of," and variations thereof are to be interpreted as exclusive rather than inclusive. Although various embodiments in the specification are presented using "comprising" language, in other cases, it is intended that the relevant embodiments be explained and described using "consisting of" or "consisting essentially of" language.

[0180] As used herein, unless otherwise indicated, the term "about" means a variability of 10% (±10%) relative to a given reference.

[0181] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

[0182] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the disclosure, which provides a general guide for those skilled in the art to many of the terms used in this application.

[0183] The term "expression" is used herein in its broadest sense and includes the production of RNA or RNA and protein. With respect to RNA, the term "expression" or "translation" particularly relates to the production of peptides or proteins. Expression can be temporary or can be stable.

[0184] As used herein, the term "NAb titer" is a measure of how many neutralizing antibodies (e.g., anti-AAV NAbs) are produced that neutralize the physiological effects of the epitope to which they target (e.g., AAV). Anti-AAV NAb titers can be measured as described in Calcedo, R et al., "Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses," Journal of Infectious Diseases, 2009. 199(3): 381-390, which is incorporated herein by reference.

[0185] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a coding sequence, a promoter, and possibly other regulatory sequences thereof, which can be delivered to a packaging host cell via a genetic element (e.g., a plasmid) and packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector contains the coding sequence for a gene product described herein, flanked by a packaging signal and other expression control sequences of the viral genome, such as those described herein.

[0186] Abbreviation " sc " refers to self-complementary. " Self-complementary AAV " refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, without waiting for the second chain synthesis mediated by the cell, two complementary half scAAVs will associate to form a double-stranded DNA (dsDNA) that is easy to replicate and transcribe immediately. See, for example, DM McCarty et al., " Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis" in gene therapy, (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0187] As used herein, the term "operably linked" refers to both expression control sequences that are contiguous to a gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest.

[0188] The term "heterologous," when used in conjunction with a protein or nucleic acid, indicates that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to one another in nature. For example, nucleic acids are often recombinantly produced, having two or more sequences from unrelated genes arranged to produce a new, functional nucleic acid. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene. Thus, the promoter is heterologous with respect to the coding sequence.

[0189] A "replication-defective virus" or "viral vector" refers to a synthetic or artificial virus particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged within the viral capsid or envelope is replication-defective; that is, it cannot produce progeny virions but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless" - containing only the transgene of interest, flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection by progeny virions will not occur unless the viral enzymes required for replication are present.

[0190] In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endonucleases and exonucleases can also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded DNA and / or double-stranded DNA and RNA. Such steps can contain a mixture of single nucleases or nucleases for different targets, and can be endonucleases or exonucleases.

[0191] The term "nuclease-resistant" indicates that the AAV capsid has been fully assembled around the expression cassette designed to deliver the transgene to the host cell and protect these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present during the production process.

[0192] In the context of the present invention, the term "translation" relates to the ribosome process whereby an mRNA chain controls the assembly of an amino acid sequence to produce a protein or peptide.

[0193] As used throughout this specification and claims, the terms "comprises" and "comprising" include other components, elements, integers, steps, etc. In contrast, the term "consisting of and its variations do not include other components, elements, integers, steps, etc.

[0194] As described above, unless otherwise stated, the term "about" when used to modify a numerical value means a variation of ±10%.

[0195] The following examples are illustrative only and are not intended to limit the invention.

[0196] Examples

[0197] The following examples report extensive deamidation of AAV8 and additional different AAV serotypes, along with supporting evidence from structural, biochemical, and mass spectrometric approaches. The extent of deamidation at each site depends on the age of the vector as well as multiple primary sequence and 3D structural factors, but is largely independent of the conditions under which the vector is recovered and purified. Deamidation is demonstrated to potentially impact vector transduction activity, with early time-point loss of vector activity associated with rapidly progressive spontaneous deamidation at several AAV8 asparagine residues. Mutational strategies to stabilize side-chain amides are explored, thereby improving vector transduction and reducing batch-to-batch molecular variability, a critical issue in biomanufacturing. This study demonstrates previously unknown aspects of AAV capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

[0198] In Example 1, we characterize post-translational modifications of the AAV8 vector capsid using one- and two-dimensional gel electrophoresis, mass spectrometry, and de novo structural modeling. After identifying multiple putative deamidation sites on the capsid surface, we evaluated their effects on capsid structure and function both in vivo and in vitro. Example 1 further extends this analysis to AAV9 to determine whether this phenomenon applies to serotypes other than AAV8, confirming that deamidation of the AAV capsid is not serotype-specific. Examples 2 to 5 illustrate deamidation in different AAVs.

[0199] Example 1: Deamidation of amino acids on the surface of adeno-associated virus capsid

[0200] A. Materials and Methods

[0201] 1D and 2D gel electrophoresis

[0202] For 1D SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, AAV vectors were first denatured at 80°C for 20 minutes in the presence of lithium dodecyl sulfate and a reducing agent. They were then run on a 4-12% Bis-Tris gel at 200 V for 90 minutes and stained with Coomassie blue. Figure 1A-1D 2D gel electrophoresis was performed by Kendrick Laboratories, Inc. (Madison, WI) for the data in the previous section. For subsequent experiments, 2D SDS-PAGE was performed in-house. For this purpose, 3 × 10 11The AAV vector of GC was combined with a 500U turbonuclease marker (Accelagen, San Diego, CA) in 150 μL of phosphate-buffered saline (PBS) containing 35 mM NaCl and 1 mM MgCl and incubated at 37°C for ten minutes. Next, nine volumes of absolute ethanol were added, the sample was vortexed, and then incubated at -80°C for at least two hours, then incubated on ice for five minutes, and then centrifuged at maximum speed at 15°C for 30 minutes. The supernatant was poured off and the pellet was air-dried, which was then resuspended in resuspension buffer #1 [ddH2O containing 0.15% SDS, 50 mM dithiothreitol (DTT), 10 mM Tris pH 7.5, and 1 μL pH 6-9 ampholytes (Thermo Fisher Scientific ZM0023), added once a day] and incubated undisturbed at room temperature. After 30 minutes, the sample tube was flicked to mix it, 1 μg of chicken conalbumin marker (Sigma Aldrich, St. Louis, MO) was added, and the sample was incubated at 37°C for 30 minutes, flicked to mix for 15 minutes. The sample was then transferred to 50°C for 15-20 minutes, vortexed, incubated at 95°C for 2.5 minutes, and allowed to cool before centrifuging at maximum speed for one minute and vortexed briefly. 10 μL of each sample was then mixed with 140 μL of resuspension buffer #2 (ddH2O containing 9.7 M urea, 2% CHAPS, 0.002% bromophenol blue, and 0.05% ampholytes, as described above, added once a day) and incubated at room temperature for ten minutes. The mixture was then applied to pH gradient (IPG) strips immobilized at pH 6-10 (Thermo Fisher Scientific, Waltham, MA) and run on a ZOOM IPG Runner system according to the manufacturer's instructions. The following isoelectric focusing parameters were used: 100-1,000 V for 120 min, 1,000-2,000 V for 120 min, and 2,000 V for 120 min, with run limits of 0.1 W and 0.05 mA per strip. The IPG strips were then reduced and loaded onto single-well 4-12% Bis-Tris gels and run for 1D as described above. The relative migration of AAV VPs was determined by comparing the internal control protein turbonuclease (Anolon, 27 kDa) to hen egg white conalbumin (Sigma-Aldrich, 76 kDa, pI 6.0-6.6).

[0203] 2. Vector Production

[0204] Recombinant AAV vectors for 1D and 2D gel electrophoresis and mass spectrometry experiments were produced by Vector Core at the University of Pennsylvania and purified by cesium chloride or iodixanol gradients as previously described. (Lock M et al., Human Gene Therapy 2010; 21(10): 1259-71; Gao GP et al., Proc. Natl. Acad. Sci. USA 2002; 99(18): 11854-9). Affinity-purified vectors were generated as follows: HEK293 cells were grown in ten 36-layer superstack containers (Corning) and co-transfected with a mixture of a vector genome plasmid (pAAV-LSP-IVS2.hFIXco-WPRE-bGH), a trans plasmid containing the AAV2 rep and AAV8 cap genes, and an adenoviral helper plasmid. PEIpro (PolyPlus) was used as the transfection reagent. Five days after transfection, the supernatant was collected, clarified by Sartoguard PES Dicap filter (Sartorius Stedim), and treated with benzonase (benzonase) (Millipore), and then salt was added to reach 0.6 M. The clarified bulk collection material was concentrated ten times by tangential flow filtration (TFF), and then diafiltered for four volumes of affinity column loading buffer. The vector was captured on POROS Capture Select (Thermo Fisher Scientific) affinity column, and the vector peak was directly eluted into neutralization buffer at low pH. The neutralized eluate was diluted into a binding buffer at high pH, ​​and loaded onto an anion exchange polishing column (Cimultus QA-8; Bia separation), where the preparation was enriched for (complete) particles containing genomes. The complete vector particles were eluted with a shallow salt elution gradient, and immediately neutralized. Finally, the vector was subjected to a second round of TFF for final concentration and buffer exchange into formulation buffer (PBS + 0.001% pluronic F-68).

[0205] Mutant vectors for in vitro assays were generated by small-scale triple transfection of HEK293 cells in six-well plates. 5.6 μL of 1 mg / mL polyethyleneimine solution was mixed with plasmid DNA (0.091 μg cis plasmid, 0.91 μg trans plasmid, 1.82 μg δF6 Ad-helper plasmid in 90 μL serum-free medium), incubated at room temperature for 15 minutes, and added to the cells along with another 0.8 mL of fresh serum-free medium. The next day, 0.5 mL of top culture medium was replaced with complete serum medium. Three days after transfection, the vectors were harvested by three freeze / thaw cycles, then centrifuged to remove cell debris and collect the supernatant. The cis plasmid contained a transgenic cassette encoding a firefly luciferase transgene under the control of a chicken β-actin (CB7) promoter, a Promega chimeric intron, and a rabbit β-globin (RBG) polyadenylation signal. The trans plasmid encodes the wtAAV8 cap gene; to generate mutant AAV8 cap variants, the Quikchange Lightning Mutagenesis Kit (Agilent Technologies, Wilmington, DE) was used. The vector was titrated as previously described. (Lock M et al., Human Gene Ther. 2010; 21(10): 1259-71).

[0206] For time course vector production experiment, vector is produced by the medium-scale triple transfection of HEK293 cells in 15cm tissue culture dishes.For each plate, 36 μ L 1mg / mL polyethyleneimine solution is mixed with plasmid DNA (0.6 μ g cis plasmid, 5.8 μ g trans plasmid, 11.6 μ g δF6 Ad-helper plasmid) in 2mL serum-free culture medium, incubation continues 15 minutes at room temperature, and on the plate renewed with 14ml serum-free culture medium, it is added to the cell with about 60% confluence. The next day, the top culture medium of 8ml is replaced with fresh full serum culture medium. By collecting all top culture mediums, cells are scraped from culture dishes and frozen at-80 ℃ to collect vector. By applying 3 freezing / thawing cycles and by centrifugal clarification lysate, crude vector is reclaimed from supernatant / cell mixture. The vector was purified and concentrated for mass spectrometry analysis by adding benzonase, 1 M Tris pH 7.5, and 5 M NaCl to the clarified lysate to a final concentration of 20 mM Tris and 360 mM NaCl. The vector was captured on a 1 ml POROS CaptureSelect affinity column and the vector peak was eluted directly into neutralization buffer at low pH. Fractions were analyzed by absorbance at 280 nm, and the most concentrated fraction was subjected to mass spectrometry analysis.

[0207] For in vivo experiments, vectors with wtAAV8 capsid or with one of six deamidation mutants were generated as previously described; the transgene cassette contained the CB7 promoter, the PI intron, the firefly luciferase transgene, and the RBG polyadenylation signal (Lock M et al., Human Gene Ther. 2010; 21(10): 1259-71).

[0208] 3. Mass Spectrometry Run / Digestion / Analysis

[0209] Materials: Ammonium bicarbonate, DTT, iodoacetamide (IAM), and 18O-enriched water (97.1% purity) were purchased from Sigma (St. Louis, MO); and acetonitrile, formic acid, trifluoroacetic acid (TFA), 8 M guanidine hydrochloride (GndHCl), and trypsin were purchased from Thermo Fisher Scientific (Rockford, IL).

[0210] Trypsin digestion: A stock solution of 1 M DTT and 1.0 M iodoacetamide was prepared. The capsid protein was denatured and reduced at 90°C for ten minutes in the presence of 10 mM DTT and 2 M GndHCl. The sample was allowed to cool to room temperature and then alkylated with 30 mM IAM for 30 minutes at room temperature in the dark. 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 in a volume that diluted the final GndHCl concentration to 200 mM. Trypsin solution was added to a trypsin to protein ratio of 1:20 and incubated at 37°C overnight. After digestion, TFA was added to a final concentration of 0.5% to quench the digestion reaction.

[0211] For the 18O water experiments, the capsid samples were first buffer-exchanged into 100 mM ammonium bicarbonate prepared in 18O water using a Zeba spin desalting column (Thermo Fisher Scientific, Rockford, IL). To ensure complete removal of water from the sample, two buffer exchanges were performed. Stock solutions of 1 M DTT and 1 M IAM were prepared in 18O water. The same denaturation, alkylation, and digestion steps were performed as described above using the 18O water reagents and buffers.

[0212] Liquid Chromatography Tandem Mass Spectrometry: Online chromatography was performed using an Acclaim PepMap column (15 cm length, 300 μm internal diameter) and a Thermo Fisher Scientific UltiMate 3000RSLC system (Thermo Fisher Scientific) coupled to a Q Exactive HF (Thermo Fisher Scientific) NanoFlex source. The column temperature was maintained at 35°C during online analysis. Peptides were separated using 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 ran from 4% B to 6% B in 15 minutes, to 10% B in 25 minutes (40 minutes total), and then to 30% B in 46 minutes (86 minutes total). Samples were loaded directly onto the column. The column dimensions were 75 cm x 15 μm ID and were packed with 2 microns of C18 medium (Acclaim PepMap). The total time for each LC-tandem mass spectrometry run was approximately two hours due to loading, introduction, and wash steps.

[0213] Mass spectrometric data were acquired on a Q Exactive HF mass spectrometer using a data-dependent top 20 approach that dynamically selected the most abundant, yet-to-be-sequenced, precursor ions from the survey scan (200-2000 m / z). Sequencing was performed by high-energy collisional dissociation fragmentation with a target value of 1e5 ions determined by predictive automatic gain control; and precursor separation was performed with a window of 4 m / z. Survey scans were acquired at 200 m / z at a resolution of 120,000. The resolution of the HCD spectra was set to 30,000 at m / z 200 with a maximum ion injection time of 50 milliseconds and a normalized collision energy of 30. The S-lens RF level was set to 50 to optimize transmission of the m / z region occupied by peptides from the digestion. Precursor ions with single, unassigned, or six and higher charge states were excluded from the fragmentation selection.

[0214] Data processing: use BioPharmaFinder 1.0 software (Thermo Fisher Scientific) to analyze all data obtained.For peptide mapping, use single entry protein FASTA database to search, wherein carbamidomethylation is set to fixed modification, and oxidation, deamidation and phosphorylation are set to variable modification.For tandem mass spectrometry, used the mass accuracy of 10ppm, high protease specificity and 0.8 confidence level. The mass spectrum identification of deamidated peptide is relatively simple, because deamidation has added+0.984Da (-OH group and-NH2 mass difference between the group) to the mass of complete molecule. Determine the deamidation percentage of specific peptide by the mass area of ​​deamidated peptide divided by the area sum of deamidated and natural peptide. Consider the quantity of possible deamidation sites, can migrate altogether at single peak place by deamidated isobaric species at different sites. Therefore, the fragment ion that is derived from the peptide with multiple potential deamidation sites can be used for locating or distinguishing multiple deamidation sites. In these cases, the relative intensities within the observed isotopic pattern can be used to specifically determine the relative abundance of the different deamidated peptide isomers. This approach assumes that the fragmentation efficiency of all isomeric species is the same and that the deamidation sites are independent. This method allows the definition of specific sites involved in deamidation as well as potential combinations involved in deamidation.

[0215] Secondary Data Processing: Secondary analysis of raw mass spectra was performed at the University of Maryland, Baltimore, MD using the following methods. All mass spectrometric analyses were performed using Peaks Studio v5.3 software (Bioinformatics Solutions, Inc.). Data refinement was performed on the raw data files using the following parameters: m / z tolerance for precursors ≤ 10 ppm, and a minimum of 2 and a maximum of 4 precursor charge states. Input spectra were sequenced de novo using the peak algorithm with a precursor ion error tolerance of 10 ppm and a product ion error tolerance of 0.1 Da. The digestion enzyme was set to trypsin, the variable modifications were set to oxidation, phosphorylation, and deamidation, and the fixed modification was set to carbamidomethylation of cysteine.

[0216] 4. Structural Analysis of AAV Capsid

[0217] AAV8 atomic coordinates, structure factors, and related capsid models were obtained from the RCSB Protein Database (PDB ID: 3RA8). Structural refinement was performed and an electron density independent of the primary amino acid sequence of AAV8 VP3 was generated for three-dimensional (3D) structural analysis of the capsid. This analysis was performed to observe the isoaspartic acid electron density in the AAV8 capsid that was not biased by the expected primary sequence of AAV8 VP3. Using the resulting structure, the four asparagine residues in the AAV8 VP3 primary sequence were modeled with N+1 glycine residues as isoaspartic acid residues, and then the AAV8 capsid structure was refined using Crystallography and NMR System (CNS) software by strictly applying an icosahedral non-crystallographic matrix using a standard refinement protocol (Brunger AT et al., Acta Crystallogr D Biol Crystallogr 1998; 54(Pt5): 905-21). The structural model of isoaspartate was obtained from the HIC-UP database, and a molecular dictionary was generated in PRODRG for structural refinement (Kleywegt GJ, Acta Crystallographica D - Biological Crystallography 2007; 63(Pt 1): 94-100). The average electron density map of the AAV8 capsid (also in CNS) was then calculated and visualized using COOT software, and the resulting model was then slightly adjusted to fit the modeled isoaspartate residue into the electron density map (Emsley P and Cowtan K, Acta Crystallographica D - Biological Crystallography 2004; 60(Pt 12Pt 1): 2126-32). This protocol was repeated to additionally model N512 in the AAV9 VP3 primary sequence using N+1 glycine (PDB ID: 3UX1). All figures were generated using COOT, PyMol, and UCSF Chimera (Emsley P and Cowtan K et al., Acta Crystallographica Section D - Biological Crystallography 2004; 60(Pt 12Pt 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).The structures of several previously identified deamidated proteins (PDB IDs: 1DY5, 4E7G, 1RTU, 1W9V, 4E7D, and 1C9D) were obtained for comparison of the electron density maps of their deamidated isoaspartate residues with modeled isoaspartate residues from AAV8 and AAV9 (Rao FV et al., Chem Biol 2005; 12(1): 65-76; Noguchi S et al., Biochem 1995; 34(47): 15583-91; Elliott et al., J Mol Biol 2000; 297(3): 713-32).

[0218] The temperature coefficients of the deamidated residues were determined by averaging the temperature coefficients for each atom of each asparagine residue reported in the atomic coordinates of the AAV8 or AAV9 crystal structures (PDB ID: 3RA8, 3UX1).

[0219] 5. Animal Studies

[0220] All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. To evaluate vector performance, eight-week-old C57BL / 6 mice were injected intravenously with 3e10 GC wtAAV8 or capsid mutant vectors in a volume of 100 μL via tail vein injection. All mice were sacrificed on day 14. To evaluate luciferase expression in vivo, mice (approximately 20 g) were anesthetized and injected intraperitoneally with 200 μL or 15 mg / mL luciferin substrate (Perkin Elmer, Waltham, MA). Mice were imaged five minutes after luciferin administration and imaged using the IVISXenogen in vivo imaging system. The signals in the described regions of interest were quantified using Living Image 3.0 software. Measurements were performed on day 7 and day 14.

[0221] 6. Evaluation of Mutant Vector Titer and In Vitro Transduction Efficiency

[0222] Vector titer was determined by qPCR against DNAseI genome. qPCR primers were ligated to the polyadenylation sequence of the packaged transgene. To evaluate vector transduction efficiency in vitro by luciferase expression, 0.9e5 Huh7 cells / well were seeded in a black-walled 96-well plate in complete DMEM (10% fetal bovine serum, 1% penicillin / streptomycin). The next day, the culture medium was removed and replaced with 50 μL of crude or purified vector diluted in complete medium. For each crude vector sample, four dilutions were tested in a 3-fold dilution series. After 48 hours, luciferin (Promega, Madison, Wisconsin) was prepared in complete medium at 0.3 μg / μL and added to the transduced cells in a volume of 50 μL. The results were read on a Biotek Clarity luminometer. The luciferase activity / GC added to the target cells was found to be constant over a wide GC range, but could become saturated at high MOIs. Therefore, the dilution series data for linearity were checked (luminescent units vs. GC), and if saturation was apparent, the highest point was excluded and the average luciferase / GC was calculated for the values ​​in the linear range of each assay for each variant. This yields transduction efficiency values. The data were normalized to simplify comparisons by setting the value of the wt control to 1.

[0223] 7. Biodistribution

[0224] DNA was extracted from liver samples using the QIAamp DNA mini kit (Qiagen, Hilden, Germany), and then analyzed by real-time PCR for vector GC using primer / probe sets designed against the RBG polyadenylation signal of the transgene cassette as previously described (Chen SJ et al., Hum Gene Ther Clin Dev 2013; 24(4): 154-60).

[0225] Primer sequences for AAV8 mutants

[0226]

[0227]

[0228]

[0229]

[0230] B. Results

[0231] AAV8 shows substantial charge heterogeneity in its capsid protein

[0232] To quantitatively assess the presence of post-translational modifications on the AAV8 vector capsid that may affect vector performance, AAV8 total capsid protein purified by iodixanol gradient was analyzed by both 1D and 2D gel electrophoresis. In 1D reducing sodium dodecyl sulfate SDS gels, VP1, VP2, and VP3 resolved as single bands at the appropriate molecular weight ( Figure 1B )(Rose JA et al., J. Virol. 1971;8(5):766-70). When proteins are separated by charge ( Figure 1C ), each of the capsid proteins was further resolved into a series of distinct spots with different isoelectric points (pIs) ranging from pH 6.3 to >7.0 ( Figure 1D Individual spots on each VP are separated by discrete intervals of 0.1 pI units, as measured by migration relative to an internal isoelectric point standard for carbonic anhydrase isoforms, indicating that single residue charge changes have occurred. The existence of these isoforms suggests that each VP has likely undergone numerous modifications, resulting in its differential migration under isoelectric focusing.

[0233] Deamidation in which a portion of the side chain amide group (usually asparagine) is converted to a carboxylic acid Figure 1A ) is a common source of charge heterogeneity in protein preparations. To determine whether deamidation might be responsible for the different populations of VP charge isoforms, two AAV8 asparagine residues were individually mutated to aspartic acid. These capsid mutations should shift the charge by an amount equivalent to fully deamidating a single additional asparagine residue. 2D gel analysis of the mutants showed that the major sites for VP1, VP2, and VP3 were shifted to one site position (0.1 pH unit) more acidic than the equivalent sites in wild-type (wt) AAV8 ( Figure 1E-1G The magnitude of this shift is equal to the spacing observed between wtVP charge isoforms. Thus, the 2D gel patterning of AAV capsid protein is consistent with multisite deamidation.

[0234] Spontaneous deamidation occurs on the AAV8 vector capsid

[0235] To identify the modifications responsible for the discrete dot-like pattern of each capsid protein, a panel of AAV8 vectors was analyzed by mass spectrometry. The average coverage of the AAV8 capsid protein was >95% of the total VP1 sequence (data not shown). Extensive deamidation of a subset of asparagine and glutamine residues was detected by mass spectrometry, showing an increase of approximately 1 Da in the mass of the observed individual peptides compared to the predicted values ​​based on the DNA coding sequence; this pattern of deamidation was observed in all preparations of AAV8 vectors ( Figures 2A-2D ).

[0236] To assess the overall heterogeneity of deamidation between commonly used purification methods and to examine deamidation in unique regions of VP1 and VP2, nine batches of AAV8 produced in 293 cells by triple transfection were selected and purified by cesium chloride gradients, iodixanol gradients, or affinity chromatography. The vectors also differed in their promoters and transgene cassettes. To determine whether the presence of the vector genome had an effect on deamidation, AAV8 preparations produced in 293 cells by triple transfection in the absence of the cis plasmid (generating only empty capsids) were also evaluated and purified by iodixanol gradients.

[0237] There is a wide range of deamidation at asparagine and glutamine residues across the AAV8 capsid, ranging from undetectable to over 99% of individual amino acids being deamidated ( Figure 2E ). The highest level of deamidation occurred at asparagine residues (>75%) where the N+1 residue was glycine (i.e., NG pair) (Table 1). Lower levels of deamidation were detected at other asparagine residues where N+1 was not glycine (i.e., up to 17%). The average deamidation of asparagine was largely consistent between preparations. Deamidation was also detected at glutamine residues, but at a lower frequency than at asparagine; the highest percentage observed at Q467 was <2% (Figure 7). This observation was not consistent across all preparations (data not shown). The greatest inter-preparation variation was observed at residue N499 (where the N+1 residue was asparagine), with values ​​ranging from <1% to over 50% deamidation. Regardless, the variations observed in deamidation between vector preparations did not appear to be correlated with the purification method, transgene identity, or the presence of a vector genome, suggesting that these factors do not affect the deamidation rate.

[0238] Table 1: Identity of AAV8 deamidated residues of interest. Asterisks indicate residues selected for further analysis.

[0239]

[0240] Next, a series of experiments were performed to determine whether sample treatment contributed to the observed deamidation levels in AAV8. Extreme temperature (70°C for 7 days) or pH (pH 2 or pH 10 for 7 days) did not significantly induce additional deamidation in the AAV8 capsid ( Figure 4A and Figure 4B Given this resistance, it is considered unlikely that the observed deamidation occurs only during the relatively short and mild purification phase. Attempts to perform mass spectrometry analysis of unpurified vector to determine the extent of deamidation before and after purification were unsuccessful. Similarly, a heavy water control indicated that the treatments specific to the mass spectrometry workflow did not contribute to additional deamidation events ( Figure 4C ).

[0241] To validate the mass spectrometry workflow, two recombinant proteins that had been previously evaluated for deamidation were examined; the findings ( Figure 5A and Figure 5B ) is consistent with published results [Henderson, LE, Henriksson, D, and Nyman, PO (1976), "Primary structure of human carbonic anhydrase C," J. Biol. Chem., 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," ​​J. Biol. Chem., 278: 15142-1515]. In addition, a secondary institution was hired to evaluate the raw data from AAV8. This independent analysis identified the same sites as those that were deamidated, with minimal variation in the extent of modification at each site, attributed to software-to-software variations in peak detection and area calculations ( Figure 6 ).

[0242] Structural topology, temperature coefficient, and identity of N+1 amino acids contribute to deamidation frequency

[0243] Since the structure of AAV8 has been solved and published (PDB identifier: 2QA0) (Nam HJ et al., J. Virol. 2011;85(22):11791-99), the AAV8 capsid structure was next examined to provide evidence for favorable conditions for non-enzymatic deamidation and to correlate the deamidation percentage with established structural features (Nam HJ et al., J. Virol. 2007;81(22):12260-71). We focused only on asparagine residues because the factors affecting asparagine deamidation are better characterized in the literature and asparagine deamidation events are far more common than glutamine deamidation events (Robinson, NE and Robinson, AB (2001), "Molecular clocks," Proc. Natl. Acad. Sci. USA 98:944-949). The temperature (or B) coefficient for each of these residues from the AAV8 crystal structure was also determined; the temperature coefficient is a measure of the displacement of an atom from its average position, with larger values ​​indicating larger displacements, larger thermal vibrations, and therefore greater flexibility (Parthasarathy S and Murphy MR, Protein Science: A Publication of the Protein Society 1997; 6: 2561-7). Most of the asparagines of interest are located in or near surface-exposed HVRs (Table 1), which structurally favor deamidation and provide an environment for solvent exposure (Govindasamy L et al., J Virol 2013; 87(20): 11187-99). It was found that, on average, residues located in these flexible loop regions, such as β strands and α helices, were more frequently deamidated than residues in less flexible regions. For example, the NG residue at position N263, which is part of HVR I, has a high temperature coefficient and is deamidated by >98% on average ( Figure 7A and Figure 6 , Table 1). Approximately 85% of N514 was deamidated within the time (Figures 3 and Figure 6, Table 1) is also present in the HVR with an N+1 glycine (HVR V); however, the local temperature coefficient is relatively low compared to that of N263, which is due to its interactions with residues on other VP monomers at the three-fold axis. Unfavorable +1 residues and lower local temperature coefficients are associated with lower deamidation, even for HVR residues. For example, N517 is only 4% deamidated on average (Table 1); this residue has a temperature coefficient comparable to that of the highly deamidated N514, but its N+1 residue is a serine, which reduces the likelihood of deamidation events due to steric hindrance. This suggests that although the identity of the +1 residue is clearly the most influential factor, many factors cumulatively determine the extent of deamidation at a given capsid position.

[0244] To test the role of the +1 residue in asparagine deamidation, mutant vectors were generated in which the AAV8 NG site was mutated to alanine or serine at the +1 position, respectively. Model peptide studies have shown that the deamidation half-life of NG peptides is as short as 1 day, while the deamidation of NA peptides or NS peptides is generally 25-fold or 16-fold slower, respectively (Robinson NE and Robinson AB, Proceedings of the National Academy of Sciences of the United States of America 2001; 98(8): 4367-72). Mass spectrometry analysis of vector mutants confirmed that the +1 site plays a central role in determining the extent of vector deamidation. When the +1 site was changed to alanine (<5% deamidation) or serine (<14% deamidation), the NG site in this collection (>80% deamidation, in wt) showed selective stabilization of the adjacent asparagine (Table 2).

[0245] Table 2: Degree of deamidation (%) at the five AAV8 NG sites and six +1 site mutants in wt

[0246] Position / Variant WT (average) G58S G58A G264A G386S G386A G515A N57 81.8 8.4 1.9 89.7 89.7 91.6 93.6 N263 99.3 98.2 98.9 4.8 100.0 94.5 97.2 N385 89.1 96.3 94.8 97.1 13.5 2.5 97.0 N514 85.2 100.0 98.0 98.8 100.0 100.0 2.2 N540 84.5 95.0 92.6 97.9 96.9 86.1 89.5

[0247] In the intact, fully assembled AAV8 capsid, residues that are at least partially buried and less exposed to solvent and / or located in regions of low local flexibility are less frequently deamidated than residues located in more favorable environments (Table 1). Nevertheless, a few residues under unfavorable conditions are still deamidated. For example, N630 is at least partially buried but still has a detectable degree of deamidation. The presence of phenylalanine as the N+1 residue for this residue suggests that this region may be a novel site for non-enzymatic autoproteolytic cleavage within the AAV8 VP3 protein.

[0248] Structural modeling of AAV8 VP3 confirms the deamidation event

[0249] To provide direct evidence for 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 available crystal structures of this serotype (i.e., The resolution of ) is insufficient to identify the terminal atoms in the R groups and, therefore, to distinguish directly between asparagine, aspartic acid and isoaspartic acid residues. Other aspects of the structure of the aspartic acid isomers formed under these conditions provide The structure determines the chance of deamidation. This analysis is based on two assumptions: 1) the main product of spontaneous deamidation of asparagine is isoaspartic acid rather than aspartic acid, which is produced in a ratio of 3: 1 (Geiger T and Clarke S, Journal of Biological Chemistry 1987; 262 (2): 785-94); and 2) asparagine or aspartic acid can be distinguished from isoaspartic acid due to the shorter length of the electron density map corresponding to the R group of isoaspartic acid. After the succinimidyl intermediate is resolved during the deamidation reaction, when the β carbon of the R group from isoaspartic acid is incorporated into the main chain of the AAV8VP3 capsid protein backbone and lost, this will produce a shorter R group.

[0250] The AAV8 structure itself was first refined to generate AAV8 capsid electron density that was not biased by the known AAV8 VP3 sequence. The refined AAV8 crystal structure was then examined for evidence of deamidation based on the presence of shorter R groups relative to isoaspartic acid. Figure 3A-Figure 3E The electron density map confirmed that no deamidated asparagine was detected by mass spectrometry at 410 ( Figure 3B ), at position 263 ( Figure 3C )、385 places (not shown), 514 places ( Figure 3D ) and 540 ( Figure 3E ) The R group of the highly deamidated N+1 glycine residue is shorter. Therefore, the deamidation indicated by the electron density map is consistent with the data of >75% deamidation at these sites by mass spectrometry. The resulting isoaspartic acid model is comparable to the isoaspartic acid residues observed in the crystal structures of other known deamidated proteins, which supports the validity of the analysis of AAV8 (Rao FV et al., Chemical Biology 2005; 12(1): 65-76; Noguchi S et al., Biochemistry 1995; 34(47): 15583-91; Elliott et al., Journal of Mol Medicine 2000; 297(3): 713-32). This structural analysis serves as an independent confirmation of the deamidation phenomenon observed when analyzing the AAV8 capsid by mass spectrometry.

[0251] Deamidation of the AAV capsid is not serotype-specific

[0252] Serotypes other than AAV8 were investigated to demonstrate capsid deamidation. Using 2D gel electrophoresis ( Figure 11A ) and mass spectrometry ( Figure 11B ) examined AAV9 vector preparations, including controls for potential vector treatment effects ( Figures 11D-11F ). The pattern and extent of AAV9 deamidation were similar to those of AAV8. All four AAV9 NG sites were >85% deamidated; 13 non-NG sites were deamidated to a lesser extent, with a few sites showing large batch-to-batch variability in % deamidation. Next, a structural analysis workflow was applied and existing AAV9 crystallographic data were refitted ( Figure 11C , Table 3). As with AAV8, isoaspartic acid fits better into the electron density of several NG sites in the AAV9 crystal structure. 2D gel analysis (data not shown) and mass spectrometry (summarized in Table 4) were extended to five additional evolutionarily distinct serotypes (rh32.33, AAV7, AAV5, AAV4, AAV3B, and AAV1). All capsids examined contained similar deamidation patterns and extents, suggesting that this modification is widespread among clinically relevant AAV vectors and is determined by similar underlying primary sequence and structural factors.

[0253] Table 3. Identity of AAV9 deamidated residues of interest.

[0254] Conserved asparagine residues with homologous N+1 residues (compared to AAV8) are indicated in italics (determined from alignment of the full-length amino acid sequences of AAV8 and AAV9 VP1).

[0255]

[0256]

[0257] Table 4. Degree of deamidation observed in different serotypes

[0258]

[0259] Deamidation events may affect capsid assembly and transduction efficiency

[0260] One way to test the functional impact of deamidation is to replace asparagine with aspartic acid by genetic mutation. Aspartate-mutant vectors encoding a luciferase reporter gene were generated for each deamidated AAV8 asparagine by small-scale triple transfection of 293 cells and titrated by qPCR against DNAseI genomic copies ( Figure 8A). Relative to wtAAV8, the mutations minimally affected capsid assembly and were limited to the largely buried non-NG sites that have low deamidation in the wt vector. Next, the in vitro transduction efficiency of the mutant group in human liver-derived Huh7 cells was evaluated ( Figure 8B Several mutants showed impaired transduction efficiency, with positions N57, N94, N263, N305, Q467, N479, and N653 exhibiting >10-fold loss of transduction. A similar number of AAV9 sensitive sites ( Figure 11G and Figure 11H ). Typically, only a subset of residues at a given position are endogenously deamidated, so this approach may overestimate loss of function in proteins such as capsids where functional units are assembled homomonomerically; endogenous modifications at one capsid site may be compensated by adjacent subunits with intact residues. Nevertheless, it is believed that the described approach can help prioritize deamidated residues for further monitoring during production or mutation stabilization. Functional data from a population of endogenously deamidated carriers will be needed to place such loss-of-function mutagenesis data in the proper context.

[0261] Loss of carrier activity over time is associated with progressive deamidation

[0262] Given the short half-life of NG deamidation, it was thought that vector samples with an age difference of only 1 day might show 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 5 days of incubation to produce the vector and 1-2 days for vector purification. To approximate this process, a medium-scale triple transfection of 293 cells (10×15 cm per cell culture dish) was prepared with wtAAV8. The vectors were collected at 1-day intervals (2×15 cm cell culture dishes / day) for 5 days, thereby preserving the vectors by freezing them at -80°C until the end of the 5-day time point. Next, the crude vector titer and in vitro transduction efficiency were assessed as described above. As expected, the number of assembled DNAseI-resistant genomic copies increased over time ( Figure 9A Then, crude vectors at early (days 1 and 2) and late (day 5) time points were rapidly processed 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 ). The efficiency of the vector on day 5 in terms of transgene expression per GC added to the target cells was only 40% of that of the material on day 1. A decrease in this activity was also observed for the crude material, suggesting a change in the molecular composition prior to purification (Figure). A similar trend in the loss of activity of AAV9 was observed over 5 days, with vector potency decreasing by approximately 40% ( Figures 11I-11K ).

[0263] Next, deamidation of the time course samples was measured by mass spectrometry. NG site deamidation proceeded substantially within each interval, with an average of 25% deamidated on day 1 and >60% of sites converted by day 5 ( Figure 9C Deamidation of non-NG sites generally proceeded within 5 days, although levels were much lower and less consistent between days 2 and 5 ( Figure 9D Our data correlate endogenous vector deamidation with an early time-point decay in specific activity and highlight potential opportunities to capture more active vectors by shortening production cycles or finding capsid mutations that stabilize asparagine.

[0264] It should be noted that Figure 2A-2E The material used for mass spectrometry analysis in the Figures was at least 7 days post-transfection, due to the additional 2 days spent on purification. The higher NG site deamidation (>80%) in these samples suggests that deamidation likely continues at approximately the same rate after the expression period and during the recovery and purification process until the NG site is completely deamidated or the vector sample is frozen. Therefore, deamidation is largely determined by the age of the vector, rather than being a process unique to or caused by the recovery and purification process. This is illustrated by the much lower deamidation values ​​in the day 1 material compared to the day 5 material (both affinity purified).

[0265] Stabilizing NG-asparagine can improve carrier performance

[0266] Given the correlation between vector NG deamidation and loss of transduction efficiency, it was considered that stabilizing NG amide by +1 site mutagenesis could improve vector function. Vectors were generated on a small scale for AAV8 NG site mutants in which each +1 residue was converted to alanine or serine. Figure 10A ) and transduction efficiency ( Figure 10B ), single + 1 mutants are well tolerated. The G386 substitution (Aydemir F et al., Journal of Virology 2016 July; 90(16): 7196-204), which is located near the previously defined "dead zone" on the capsid surface, has an in vitro transduction defect. The loss of function of the G386 mutant may indicate a preference for the deamidated asparagine at N385. Alternatively, the additional side chain entity at the + 1 position may have a negative impact on the function independent of amide group stabilization. Although its adjacent asparagine has significant stability, the single site mutant did not significantly improve in vitro transduction (Table 2). Because the transduction activity in vitro and in vivo may be inconsistent, a subset of single site + 1 mutants for liver transduction was tested in C57BL / 6 mice. Intravenous tail vein injection (n = 3 to 5) was performed, and luciferase expression was examined by weekly imaging for 2 weeks ( Figure 10CThe in vivo and in vitro transduction data were consistent with (i.e., within) the relative error range for each assay. The G386 substitution was defective in transduction, whereas +1 site mutations at other positions were largely tolerated, transducing the liver at levels equal to, but not exceeding, wtAAV8.

[0267] Because amide stabilization at any one NG site may be necessary but not sufficient to restore function, vector variants with combinations of +1 site alanine substitutions were evaluated next. All three AAV8 NG sites (N263, N514, and N540) were recombined with a highly functional +1 alanine. Some combinations containing the triple mutant G264A / G515A / G541A assembled poorly and were dysfunctional for transduction. However, two pairwise combinations involving N263 (G246A / G515A and G264A / G541A) both improved in vitro transduction efficiency (2.0-fold and 2.6-fold that of wtAAV8, respectively) without loss of potency ( Figure 10D Because these mutations introduce at least two changes (N-amide stabilization and +1 residue side chain substitution), these data cannot conclusively link NG deamidation to loss of function. However, the data are consistent with a model established in time-course studies in which NG site deamidation may affect in vitro transduction efficiency.

[0268] Functional asparagine substitution improves batch-to-batch reproducibility of vector preparation

[0269] Another potentially problematic aspect of the reported vector deamidation profile is the high batch-to-batch variability in deamidation at some positions. For wtAAV8, this variability was most pronounced for N459 (observed deamidation ranged from 0% to 31%) and N499 (observed deamidation ranged from 0% to 53%). Variability in post-translational modifications is often practically avoided during biologic development by completely avoiding clones that exhibit this variability, carefully monitoring and controlling production strains and conditions, or performing protein engineering on affected candidates.

[0270] Since it is not possible to identify the production or processing factors that cause the variability in deamidation of N459 and N499 ( Figure 2E ), so functional amino acid substitutions were sought at these positions. We first evaluated the possibility of conservative substitution of glutamine at each position in a small-scale vector preparation. Both N459Q and N499Q were efficiently assembled into the vector and were comparable to the in vitro transduction efficiency reference of wtAAV8 ( Figure 7ANext, the 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 glutamine at positions 459 or 499 was observed in these mutants (data not shown). Liver transduction of these mutant batches was evaluated in vivo after tail vein injection in C57BL / 6 mice as described above ( Figure 7B and Figure 7C The wtAAV8 vector batch used as a control in this experiment was deamidated by 16.8% at N499, but no deamidation was detected at N459 (data not shown). Liver transduction by both mutants at day 14 was equivalent to that of wtAAV8. This data demonstrates the potential of protein engineering approaches to address deamidation-related molecular variability in manufactured AAV vectors.

[0271] C. Discussion

[0272] Non-enzymatic deamidation of asparagine and glutamine residues on the AAV8 capsid was identified and evaluated by 2D gel electrophoresis, mass spectrometry, de novo protein modeling, and both in vitro and in vivo functional studies. Deamidation has been shown to occur in a wide variety of proteins and significantly affect the activity of biological agents, 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 Immunol Clin Immunol 2016; 23(5): 396-402). Other viral proteins, such as the VP6 protein of rotavirus, have been shown by mass spectrometry to undergo deamidation events (Emslie KR et al., Funct Integr Genomics 2000; 1(1): 12-24).

[0273] The occurrence of these deamidations in AAV8 suggests that they are the result of spontaneous, non-enzymatic events. Asparagine residues are known to be more extensively deamidated than glutamine residues; the amino acids downstream of asparagine substantially influence the rate of deamidation, with N+1 glycine (ie, NG) being most efficiently deamidated. The observation that the role of the N+1 amino acid in the deamidation of the AAV capsid is clearly demonstrated, as each NG present in VP1 is deamidated at a level of >75%, while the deamidation of any of the other asparagine or glutamine in the capsid is never >20%. In fact, all NG motifs in AAV8 and AAV9 capsids (ie, 7 / 9) are also present on the surface of the capsid contained in the HVR region associated with conformational flexibility and a high rate of thermal vibration. This is consistent with previous reports of NG motifs in other proteins, which are located in regions where flexibility may be required for proper protein function, rather than in more ordered structures such as α-helices or β-sheets (Yan BX and Sun YQ, J Biol Chem 1997; 272(6): 3190-4). The preference for NG motifs in surface-exposed HVRs further enhances the deamidation rate by providing solvent accessibility and conformational flexibility, thereby promoting the formation of succinimidyl intermediates. As expected, the unfavorable environment results in a much lower deamidation rate.

[0274] An important question about the biology of AAV and its use as a vector is the functional consequences of these deamidations. Mutagenesis of the capsid DNA to convert asparagine to aspartic acid allows for the evaluation of capsids in which all amino acids at a particular site are represented as aspartic acid. However, there is no easy strategy to prevent deamidation using mutagenesis, except for potentially mutating the N+1 residue, which is confused with the direct consequences of second-site mutations. A limited number of variants have been studied in which asparagine residues were converted to aspartic acid by mutagenesis. Functional analyses include capsid assembly and in vitro and in vivo transduction. The most fundamental effect of mutagenesis on vector function is the effect of asparagine residues that are not fully deamidated at baseline and have no exposed surfaces. However, surprisingly, mutagenesis of the highly deamidated asparagine at 514 to aspartic acid does have some effect on function. This result suggests that the presence of residual amounts of the corresponding amide may affect function. This may be due in part to the presence of a hydrogen bonding interaction between N514 and D531 of another three-fold related VP3 monomer (identified in the wtAAV8 crystal structure), which is lost upon conversion of this residue to aspartic acid following deamidation.

[0275] A better understanding of the factors that influence the extent of deamidation in AAV vectors is important when assessing the impact of these deamidations on the development of novel therapeutics. Incubating the vector under extreme conditions (known to significantly accelerate deamidation kinetics) had little effect. Combined with isotope incorporation studies, this result suggests that deamidation occurs during capsid assembly and is not an artifact of vector processing or mass spectrometry analysis. Deamidation at the NG site is unlikely to have a substantial impact on vector performance because the reaction was virtually complete in every sample evaluated. However, initial functional studies suggest that residual amounts of undeamidated asparagine can contribute to function. More attention is being paid to sites where deamidation is incomplete, which in most cases is also associated with sample-to-sample variability. An example is the asparagine at position 499, which shows a deamidation range of 0% to 53%, with an average of 17%. Subtle differences in vector production conditions may contribute to this heterogeneity. The striking similarity of deamidation in AAV8 and AAV9 suggests that this is a property of this entire family of viruses.

[0276] In summary, a great deal of heterogeneity has been found in the primary amino acid structure of the AAV8 and AAV9 capsid proteins. These studies may impact the development of AAV as a vector in several ways. First, the actual amino acid sequence of the VP protein is not predicted by the corresponding DNA sequence. Second, various aspects of the production process may lead to differences in deamidation and corresponding changes in vector function. Until the factors that influence the rate of deamidation at non-NG sites are understood and its functional consequences are better understood, it may be necessary to include deamidation in the characterization of clinical-grade AAV vectors. 2D gel electrophoresis can provide an overall assessment of net deamidation, although mass spectrometry is essential for assessing deamidation at specific residues.

[0277] Example 2: Deamidation of AAV5.5.9

[0278] The novel sequences of AAV5.5.9 are provided in SEQ ID NOs: 9 and 10, respectively. Deamidation of the AAV5.5.9 vector was assessed as described for AAV9 in Example 1. Highly deamidated residues were seen at N57, N319, N442, and N502.

[0279]

[0280]

[0281] Example 3: Deamidation of AAVrh79 (clade E)

[0282] AAVrh79 was isolated from DNA extracted from the small intestine of rhesus macaques. It has been phylogenetically characterized as belonging to clade E ( Figures 14A-14D). The sequence thereof is provided herein, wherein the nucleotide sequence is in SEQ ID NO: 1, and the amino acid sequence is in SEQ ID NO: 2. Figure 14A An alignment of the amino acid sequences of AAVrh79, AAVrh.10, and AAVhu.37 is provided in . Figures 14B-14D An alignment of the nucleic acid sequences of AAVrh79, AAVrh.10, and AAVhu.37 is provided in .

[0283] AAVrh79 has three amino acid differences in its primary sequence. Whereas AAVhu37 has an Ala at position 67 and a Lys at position 169 located in its primary VP1 sequence, AAVrh79 has a glutamic acid (E) at position 67 and an Arg at position 169. Figure 11B The differences in the DNA sequence of VP1 between rh.79, hu.37 and hu.40 are shown in FIG. Vectors expressing eGFP based on various clade E variants were prepared and their relative infectivity on Huh7 cells was evaluated ( Figure 11C C57BL / 6 mice were injected with two dose levels (3×10 10 and 3×10 11 GC / mice) were injected with AAV8 or AAVrh.79 vectors expressing eGFP, and infectivity was assessed by fluorescence microscopy (data not shown).

[0284] AAVrh79-based vectors were prepared using known production techniques using the AAVrh79 nucleotide sequence for cap generation, as previously described for AAV8 vectors. Figures 15A-15B and Figure 16 The results of the production productivity and production purity evaluation are provided in Figure 2, respectively.

[0285] To evaluate the expression level using AAVrh79 containing a marker gene (firefly luciferase), 3 × 10 11GC / mice. ffLuc expression was visualized by whole-body bioluminescence imaging as previously described [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.] Figures 17A-17D The results are provided in .

[0286] In male and female cynomolgus monkeys, 10 13 GC / kg AAVrh79, the expression of AVV8 triple, AAVhu68, AAV9, AAV8 and AAVrh79 vectors was compared ( Figure 17E ).

[0287] The vector expressing the secreted transgene (201Ig IA) was injected intramuscularly into the gastrocnemius muscle of male RAG KO mice (n=5 / group) (3×10 10 or 3×10 11 GC / mouse). The results showed that after IM injection, AAV8 triplet expressed better and at the lower doses tested, the difference in expression from AAV8 triplet was quite large. At the higher doses, AVVrh79 expressed at levels comparable to the other vectors tested ( Figure 17F ).

[0288] Pre-screening of NAbs ( Figure 18A ), and injected with AAVG2.TBG.eGFP.WPRE.bGH (1×10 13 ddGC / kg, intravenously). Animals were euthanized 7 days after treatment and autopsied to isolate liver and other tissues for analysis. GFP expression in liver and spleen was assessed on day 7 ( Figure 18B and 18C ).exist Figure 18D The vector levels detected in various tissues are shown in .

[0289] Various AAV8 and AAVrh79 vectors were produced, and in some cases, multiple batches were produced. The yields of these AAV9 and AAVrh79 vector batches were compared ( FIG19 ).

[0290] The deamidation of the AAVrh79 vector was evaluated as described for AAV8 and AAV9 in Example 1. The results showed that the vector contained four highly deamidated amino acids (N57, N263, N385, N514), which correspond to asparagine in an asparagine-glycine pair based on the numbering of AAVrh79 (SEQ ID NO: 1). Lower deamidation percentages were consistently observed at residues N94, N254, and N410.

[0291]

[0292]

[0293] Example 4: Preparation of AAV8.2.08

[0294] As discussed in WO 2017 / 180854 (incorporated herein by reference), several AAV8 mutants c41, c42, c46, g110, g113, g115, and g117 were generated that have mutations in the HVR.VIII region. As discussed by Gurda et al., the primary ADK8 epitope is located in the HVR.VIII region (amino acids 586 to 591 using AAV8 vp1 numbering). These mutants were tested for ADK8 resistance in vitro, and some of them were tested for ADK8 resistance in vivo. See, for example, Lochrie 2006, cited above.

[0295] AR2.1-9 was randomly picked. AR2.25-61 was selected based on frequency. Randomly picked variants and variants with higher frequencies were all viable in terms of 6-well plate yield and Huh7 transduction. Figure 24 Expression in various tissues of AAV8.AR2.08 is shown (leftmost set of bars).

[0296] Example 5: Deamidation of AAV8.AR2.08

[0297] The novel sequences of AAV8.AR2.08 are provided in SEQ ID NOs: 17 and 18, respectively, which were designed as in Example 4.

[0298] A. A modified AAV8.AR2.08 vector was generated and evaluated for modification as described for AAV8 in Example 1. The results show that the vector contains five highly deamidated amino acids (N57, N263, N385, N514, and N540), which correspond to asparagine in an asparagine-glycine pair based on the numbering of AAV8.AR2.08 (SEQ ID NO: 18). A consistently lower percentage of deamidation was observed at residues N94, N254, and N410. In contrast to AAV8, no deamidation was observed at positions N459 (average 7% in AAV8) or N499 (average 17% in AAV8).

[0299]

[0300]

[0301] B. Single-cell RNA-seq reveals tissue localization and transcriptional signatures of transduced hepatocytes isolated from nonhuman primates after treatment with AAV8.

[0302] Single-cell RNA sequencing has proven to be a powerful technique for characterizing the cellular transcriptome with unprecedented single-cell resolution. In the present work, single-cell RNA-seq was used to investigate the transcriptional landscape of primary hepatocytes isolated from rhesus macaques following treatment with an AAV8 vector expressing GFP. Transcriptome analysis of FACS-sorted GFP+ and GFP- cells revealed the tissue localization of transduced cells within the liver lobule, as well as genes and regulatory pathways involved in hepatocyte transduction and regulation of transgene expression.

[0303] For the study design, rhesus monkeys were treated with 1×1013 ddGC / kg AAV8.TBG.EGFP.WPRE (n=1) or 1×1013 ddGC / kg AAV8.2.08.TBG.EGFP.WPRE (AAV8 variant, n=1). Animals were euthanized 7 days after treatment and necropsy was performed to isolate the liver from both animals. After collagenase treatment and gradient centrifugation, isolated hepatocytes were FACS sorted onto BD Precise TM 96-well plates. 192 single cells (96 GFP+ and 96 GFP-) were isolated from each animal and then used to prepare single-cell RNA-seq libraries according to the standard BD Precise™ protocol. Data were analyzed using the Seurat, Scran, and Scater packages in R to determine the transcripts differentially expressed between GFP- and GFP+ sorted cells, and the established transcriptional expression signatures were used to spatially reconstruct the isolated cells within the liver lobule.

[0304] AAV8.AR2.08 was found to have increased liver tropism compared to AAV8 and exhibited a 1.5-fold increase in transduction efficiency. Single-cell transcriptome analysis of sorted hepatocytes revealed that transgene-expressing cells were evenly distributed throughout the liver lobules, showing a slight preference for the portal region, which was also observed in histopathology. Interestingly, a subpopulation of sorted GFP- cells was found to express transgene transcripts at levels comparable to sorted GFP+ cells, indicating that despite the absence of detectable levels of translated protein, these cells had in fact been transduced and were expressing transgene mRNA. Comparison of the transcriptional profiles of GFP- and GFP+ cells revealed differentially expressed transcripts involved in viral mRNA translation, thereby elucidating possible pathways involved in the regulation of transgene protein expression in transduced cells.

[0305] C. Isolation of AAV-8 Variants with Better Liver Transduction and Higher Liver Specificity in Nonhuman Primates Using Directed Evolution in a Human Liver Xenograft Model

[0306] To obtain AAV variants with better transduction and higher specificity, AAV directed evolution (a benchmark for liver gene therapy) was performed using saturation mutagenesis targeting surface-exposed sites on the AAV8 capsid, followed by two rounds of in vivo enrichment in a human liver xenograft mouse model, and an AAV8 variant called AAV8.2.08 was isolated. After intravenous injection into non-human primates at a dose of 1e13 genome copies (GC) / kg body weight, AAV8.AR2.08 delivered fewer vector genome copies in various organs (including lungs, heart, stomach, pancreas, kidneys, and mesenteric lymph nodes) than AAV8, while delivering more vector genome copies in the liver, implying better liver transduction and higher tissue specificity. Next-generation sequencing showed that the significant enrichment of AAV8.AR2.08 during in vivo selection demonstrated the potential of the method for isolating capsids with new and improved tropism. Figure 21 27 show other comparisons. Figure 28 The biodistribution of AAV8.AR2.08 and AAV8 is shown.

[0307] D. Add barcode

[0308] Six black mice were intravenously injected with 2×10 12 A mixture of rAAVG3 from 12 preparations ( Figure 18B Each preparation contains a separate barcode within the vector genome, allowing identification of the specific preparation ( Figure 18A Two weeks later, the animals were euthanized and tissues were collected. As expected, rAAVG3 expression was higher in the liver than in the heart or muscle. Figure 18CTissue distribution experiments showed that the actual frequency of barcodes in the injected vector mixture matched the theoretical frequency ( Figure 18D :total; Figure 19A 、 19B :muscle; Figure 19C 、 19D : Heart; and Figure 19E 、 19F : liver), with slight abnormalities in BC02 and BC06 ( Figures 20A-20C ). All documents cited in this specification are incorporated herein by reference. U.S. Provisional Patent Application Nos. 62 / 722,388 and 62 / 722,382, both filed on August 24, 2018, U.S. Provisional Patent Application Nos. 62 / 703,670 and 62 / 703,673, both filed on July 26, 2018, U.S. Provisional Patent Application Nos. 62 / 677,471 and 62 / 677,474, both filed on May 29, 2018, U.S. Provisional Patent Application No. 62 / 667,585, filed on May 29, 2018, and U.S. Provisional Patent Application No. 62 / 635,964, filed on February 27, 2018, are incorporated herein by reference. U.S. Provisional Patent Application No. 62 / 667,881, filed May 7, 2018, U.S. Provisional Patent Application No. 62 / 667,888, filed May 7, 2018, U.S. Provisional Patent Application No. 62 / 667,587, filed May 6, 2018, U.S. Provisional Patent Application No. 62 / 663,797, filed April 27, 2018, U.S. Provisional Patent Application No. 62 / 663,788, filed April 27, 2018, and U.S. Provisional Patent Application No. 62 / 635,968, filed February 27, 2018, are incorporated herein by reference. The SEQ ID NOs cited herein and appearing in the attached sequence listing are incorporated by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A recombinant adeno-associated virus (rAAV), comprising: (A) AAVrh79 capsid, comprising one or more of the following: (1) AAVrh79 capsid protein, the AAVrh79 capsid protein comprising: a heterogeneous population of AAVrh79 vpl proteins selected from the group consisting of vpl proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 738 of SEQ ID NO:2, vpl proteins produced from SEQ ID NO:1, or vpl proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 738 of SEQ ID NO:2 that is at least 70% identical to SEQ ID NO:1, a heterogeneous population of AAVrh79 vp2 proteins selected from the group consisting of vp2 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO:2, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2214 of SEQ ID NO:1, or vp2 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 738 of SEQ ID NO:2 that is at least 70% identical to at least nucleotides 412 to 2214 of SEQ ID NO:1, a heterogeneous population of AAVrh79 vp3 proteins selected from the group consisting of vp3 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO:2, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2214 of SEQ ID NO:1, or vp3 proteins produced from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 204 to 738 of SEQ ID NO:2 that is at least 70% identical to at least nucleotides 607 to 2214 of SEQ ID NO:1; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 138 to 738 of SEQ ID NO:2; and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 204 to 738 of SEQ ID NO:2, wherein: the vp1 proteins, the vp2 proteins, and the vp3 proteins contain a subpopulation having amino acid modifications that include at least two highly deamidated asparagines (N) of the asparagine-glycine pairs of SEQ ID NO:2, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (B) A vector genome in the AAVrh79 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product, the non-AAV nucleic acid sequence being operably linked to a sequence that directs expression of the product in a host cell.

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

3. The rAAV of any one of claims 1 or 2, wherein the capsid further comprises one or more deamidated glutamines, wherein the one or more deamidated glutamines are deamidated to (α)-glutamate, γ-glutamate, an interconverting (α)-glutamate / γ-glutamate pair, or a combination thereof.

4. The rAAV of any one of claims 1 to 3, wherein the AAVrh79 capsid comprises a subpopulation having one or more of: (a) at least 75% of the asparagine (N) in the asparagine-glycine pair at position 57 of the vp1 protein based on the numbering of SEQ ID NO: 2 is deamidated; (b) at least 75% of the N in the asparagine-glycine pair at position 263 of the vp1, v2, and vp3 proteins, based on residue numbering of the amino acid sequence of SEQ ID NO: 2, is deamidated; (c) at least 70% of the N in the asparagine-glycine pair at position 385 of the vp1, v2 and vp3 proteins, based on residue numbering of the amino acid sequence of SEQ ID NO: 2, is deamidated; and / or (d) at least 85% of the N in the asparagine-glycine pair at position 514 of the vp1, v2 and vp3 proteins based on the residue numbering of the amino acid sequence of SEQ ID NO: 2 is deamidated.

5. The rAAV of any one of claims 1 to 4, wherein the rAAVrh79 capsid comprises a subpopulation of vpl wherein 75% to 100% of the N at position 57 of the vpl protein is deamidated as determined using mass spectrometry.

6. The rAAV of any one of claims 1 to 5, wherein the rAAVrh79 capsid comprises a subpopulation of vp1, vp2, and / or vp3 proteins, wherein 75% to 100% of the N at position 263 based on numbering of SEQ ID NO: 2 is deamidated as determined using mass spectrometry.

7. The rAAV of any one of claims 1 to 6, wherein the rAAVrh79 capsid comprises a subpopulation of vp1, vp2, and / or vp3 proteins, wherein 75% to 100% of the N at position 385 based on numbering of SEQ ID NO: 2 is deamidated as determined using mass spectrometry.

8. The rAAV of any one of claims 1 to 7, wherein the rAAVrh79 capsid comprises a subpopulation of vp1, vp2, and / or vp3 proteins, wherein 75% to 100% of the N at position 514 based on numbering of SEQ ID NO: 2 is deamidated.

9. The rAAV of any one of claims 1 to 8, wherein the nucleic acid sequence encoding the protein is SEQ ID NO: 1 or a sequence encoding the amino acid sequence of SEQ ID NO: 2 that is at least 80% to at least 99% identical to SEQ ID NO:

1.

10. The rAAV of claim 9, wherein the nucleic acid sequence is at least 80% to 97% identical to SEQ ID NO: 1.

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