In vitro parvovirus capsid manufacturing

Abstract

The present disclosure relates to in vitro methods for assembling parvovirus capsids, in vitro methods for filling parvovirus capsids and in vitro methods for manufacturing filled parvovirus capsids. The present disclosure further relates to assembled parvovirus capsids, filled parvovirus capsids and compositions comprising such.

Description

[0001] IN VITRO PARVOVIRUS CAPSID MANUFACTURING

[0002] Field of the Invention

[0003] The present disclosure relates to in vitro methods for assembling parvovirus capsids, in vitro methods for filling parvovirus capsids and in vitro methods for manufacturing filled parvovirus capsids. The present disclosure further relates to assembled parvovirus capsids, filled parvovirus capsids and compositions comprising such.

[0004] Background of the Invention

[0005] Research and clinical applications of recombinant parvoviruses, such as recombinant adeno-associated virus (rAAV), has significantly increased in recent years alongside regulatory approvals of rAAV gene therapy products. To date, most rAAV vectors, as well as AAV empty capsids, are produced in eukaryotic cells. However, during the manufacturing of AAV vectors, transgene packaging is usually imperfect. The capsids that do not incorporate a transgene payload are referred to as empty capsids, which highlights the necessity of improving the proportion of capsids filled with DNA for the process of scalable vector production.

[0006] Furthermore, the three distinct viral capsid proteins (e.g., viral protein 1 (VP1), VP2, and VP3) that compose an AAV capsid each play a unique role in cell-entry and transduction, though AAV capsid assembly is stochastic, leading to a highly heterogeneous populations of capsids of variable viral capsid protein composition. For example, it has been demonstrated that even the single-most abundant viral capsid protein stoichiometry represents only a small percentage of a given manufactured AAV capsid population. This heterogeneity highlights the need for improved methods of parvovirus capsid production strategies that can enable controlled, reproducible viral capsid protein stoichiometries at large scale and low cost.

[0007] Summary of the Invention

[0008] The present disclosure relates to in vitro methods for assembling parvovirus capsids, in vitro methods for filling parvovirus capsids and in vitro methods for manufacturing filled parvovirus capsids. The present disclosure further relates to assembled parvovirus capsids, filled parvovirus capsids and compositions comprising such.

[0009] In a first aspect, the present disclosure relates to a method of in vitro assembling parvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor;b. combining the inhibited parvovirus viral capsid proteins in vitro; andc. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) invitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby assembling parvovirus capsids and providing assembled parvovirus capsids.In another aspect, the present disclosure relates to a method of in vitro filling parvovirus capsids,comprising the steps of: a. providing:i. one or more populations of assembled parvovirus capsids comprising parvovirus viralcapsid proteins in vitro; ii. a nucleic acid molecule comprising a transgene of interest and a Rep binding domainin vitro; and iii. a Rep protein in vitro;b. combining the one or more populations of assembled parvovirus capsids, the nucleic acidmolecule, and the Rep protein in vitro under filling conditions which allow filling of theassembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids and providing filled parvovirus capsids.In another aspect, the present disclosure relates to a method of in vitro filling parvovirus capsids,comprising the steps of: a. providing:i. a population of assembled parvovirus capsids comprising parvovirus viral capsidproteins in vitro; ii. a nucleic acid molecule comprising a transgene of interest and a Rep binding domainin vitro; iii. a Rep proteins in vitro; andiv. a reaction mixture in vitro;b. combining the population of assembled parvovirus capsids, the nucleic acid molecule, theRep protein and the reaction mixture in vitro under filling conditions which allow filling of theassembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids and providing filled parvovirus capsids.In another aspect, the present disclosure relates to a method of in vitro manufacturing filled parvoviruscapsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby providing one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro d. providing: i. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain in vitro; and ii. a Rep protein in vitro; e. combining the population of assembled parvovirus capsids obtained in c., the nucleic acid molecule and the Rep protein in vitro under filling conditions which allow filling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids, thereby manufacturing filled parvovirus capsids.

[0010] In another aspect, the present disclosure relates to a method of in vitro manufacturing filled parvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby providing one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro d. providing: i. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain in vitro ; ii. a Rep protein in vitro; and iii. a reaction mixture in vitro e. combining the population of assembled parvovirus capsids obtained in c., the nucleic acid molecule, the Rep protein and the reaction mixture in vitro under filling conditions which allow filling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids, thereby manufacturing filled parvovirus capsids.

[0011] In another aspect, the present disclosure relates to a method of in vitro controlling the stoichiometry of parvovirus viral capsid proteins in assembled parvovirus capsids, comprising the step of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor, wherein the one or more populations of inhibited parvovirus viral capsid proteins are provided at a defined stoichiometry; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby assembling parvovirus capsids and providing assembled parvovirus capsids with a defined stoichiometry of parvovirus viral capsid proteins.

[0012] In another aspect, the present disclosure relates to an AAV Rep fusion protein comprising: a. An AAV Rep domain or a AAV Rep protein, and b. A cleavable amino acid sequence, such as a SUMO tag.

[0013] In another aspect, the present disclosure relates to a vector encoding the AAV Rep fusion protein according to the present disclosure.

[0014] In another aspect, the present disclosure relates a method of producing the AAV Rep fusion protein according to the present disclosure, comprising the steps of: a. Transforming a prokaryotic microbial system, such as E. coli, with the vector encoding the AAV Rep fusion protein according to the present disclosure; and b. Incubating the prokaryotic microbial system, such as E. coli, thereby producing the AAV Rep fusion protein. In another aspect, the present disclosure relates a method of producing the AAV Rep fusion protein according to the present disclosure, comprising the steps of: a. Transforming an eukaryotic microbial system, such as P. pastoris or S. cerevisiae, with the vector encoding the AAV Rep fusion protein according to the present disclosure, b. Incubating the eukaryotic microbial system, such as P. pastoris, or S. cerevisiae, thereby producing the AAV Rep fusion protein.

[0015] In another aspect, the present disclosure relates an AAV VP fusion protein comprising: a. An AAV VP domain or an AAV VP protein, and b. A cleavable amino acid sequence, such as a SUMO tag.

[0016] In another aspect, the present disclosure relates a vector encoding the AAV VP fusion protein according to the present disclosure.

[0017] In another aspect, the present disclosure relates to a method of producing a method of producing the AAV VP fusion protein according to the present disclosure, comprising the steps of: a. Transforming a prokaryotic microbial system, such as E. coli, with the vector encoding the AAV VP fusion protein according to the present disclosure; and b. Incubating the prokaryotic microbial system, such as E. coli, thereby producing the AAV VP fusion protein.

[0018] In another aspect, the present disclosure relates to a method of producing the AAV fusion protein according to the present disclosure, comprising the steps of: a. Transforming a eukaryotic microbial system, such as P. pastoris or S. cerevisiae, with the vector encoding the AAV VP fusion protein according to the present disclosure b. Incubating the eukaryotic microbial system, such as P. pastoris or S. cerevisiae, thereby producing the AAV fusion protein.

[0019] In another aspect, the present disclosure relates to a fusion protein consisting of or comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35-43, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91%, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity an amino acid sequence selected from SEQ ID NOs: 35-43. In another aspect, the present disclosure relates to a fusion protein consisting of or comprising an amino acid sequence selected from SEQ ID NOs: 35-43.

[0020] Brief Description of the Drawings

[0021] Figure 1

[0022] Figure 1 shows a novel method of in vitro AAV capsid filling: (A) Expression of empty AAV2 capsids and rep proteins by suspension HEK293 cells in serum-free and antibiotic-free medium and using double plasmid transfection method. (B) Generation of plasmid containing backbone, ITR regions, CMV enhancer, CMV promoter, eGFP reporter gene, and poly-A signal. (C) Capsid quantification through ELISA, (D) In vitro capsid filling; (E) Genome copy quantification using qPCR.

[0023] Figure 2

[0024] Figure 2 shows ELISA standard curve: Capsid concentration was quantified by generating a sigmoidal curve through four Parameter Logistic (4PL) fitting. A 100-fold sample dilution was found to be in the linear range of this curve. As such, the original sample concentration was determined to be 1 .86e10 empty capsids / mL.

[0025] Figure 3

[0026] Figure 3 shows final genome copy concentrations, measured by qPCR, of processed samples and two pure plasmid controls (one treated with DNAse, the other without DNAse).

[0027] Figure 4

[0028] Figure 4 shows effect of capsid addition on plasmid digestion by DNAse. The x-axis shows the concentration of plasmid added to each condition / control (prior to capsid filling and / or DNAse treatment). This was determined by using untreated pGFP as a reference concentration and extrapolating sample concentrations based on dilution factor. The y-axis shows the final qPCR readout after DNAse treatment.

[0029] Figure 5

[0030] Figure 5 shows plasmid packaging and capsid filling efficiencies. Plasmid packing efficiency refers to the relative number of genomes that have been encapsulated; capsid filling efficiency refers to the relative number of filled capsids compared to the total number of capsids.

[0031] Figure 6

[0032] Figure 6 shows qPCR results from the different tested capsid filling condition. Figure 7

[0033] Figure 7 shows a novel method of in vitro AAV capsid filling: (A) Expression of empty AAV2 capsids and rep proteins by suspension HEK293 cells in serum-free and antibiotic-free medium and using double plasmid transfection method. (B) Generation of plasmid containing backbone, ITR regions, CMV enhancer, CMV promoter, eGFP reporter gene, and poly-A signal. (C) Capsid quantification through ELISA. (D) In vitro capsid filling. (E) Affinity chromatographic purification using AAVX. (F) Genome copy quantification using qPCR. (G) Infectious unit quantification using eGFP reporter fluorescent assay.

[0034] Figure 8

[0035] Figure 8 shows gating strategy for Hek293 cells transduced with AAV2 CMV-GFP vectors.

[0036] Figure 9

[0037] Figure 9 shows vector genome titer by qPCR.

[0038] Figure 10

[0039] Figure 10 A) shows capsid titer by AAV2 capsid ELISA. Figure 10 B) shows full capsid ratio as calculated by dividing vector genome titer (VG / mL) with capsids titer (Cap / mL).

[0040] Figure 11

[0041] Figure 11 shows a fluorescence microscopy picture. Cells transduced with Control and in vitro filled AAV2-GFP vectors and subsequently analyzed for GFP expression by fluorescence microscope.

[0042] Figure 12

[0043] Figure 12 shows infectivity. Cells transduced with Control and in vitro filled AAV2-GFP vectors and subsequently analyzed for GFP expression by flow cytometry.

[0044] Figure 13

[0045] Figure 13 shows infectivity as a function of the number of capsids (full and empty) added to the cells.

[0046] Figure 14

[0047] Figure 14 shows genome copy concentrations, measured by qPCR, for in vitro capsid filling for AAV5.

[0048] Figure 15

[0049] Figure 15 shows SDS PAGE of clarified E. coli lysate containing his-SUMO-Rep-EPEA fusion proteins. Number indicates proposed bands for his-SUMO Rep proteins based on expected MW. 1 . his-SUMO Rep40, MW -58 kDa; 2. his-SUMO Rep52, MW -70 kDa; 3. his-SUMO Rep68, MW -84 kDa; 4. His- SUMO Rep78, MW -94 kDa. Expression of IPTG induced cultures, shown as ‘+IPTG’ lanes, were compared to uninduced expression.

[0050] Figure 16

[0051] Figure 16 shows SDS PAGE of purified Rep proteins with his-SUMO fusion tag. Black arrows indicate Rep protein (Rep40, MW 40 kDa; Rep52, MW 52 kDa; Rep68, MW 68 kDa; Rep78, MW 78 kDa) + his- SUMO (MW -18 kDa).

[0052] Figure 17

[0053] Figure 17 shows SDS PAGE showing successful cleavage of SUMO after incubation with SUMO protease for Rep40 and Rep52. Black arrows indicate Rep protein (Rep40, MW 40 kDa; Rep52, MW 52 kDa; Rep68, MW 68 kDa; Rep78, MW 78 kDa) + his-SUMO (MW -18 kDa). White arrows indicate Rep40 and Rep 52 (Rep40, MW 40 kDa; Rep52, MW 52 kDa) after successful his-SUMO cleavage (SUMO band unmarked at -18 kDa).

[0054] Figure 18

[0055] Figure 18 shows SDS PAGE showing successful cleavage of his-SUMO after incubation with SUMO protease at 5°C for Rep68 and Rep78. Black arrows indicate Rep68 (Rep68, MW 68 kDa) + his-SUMO, and Rep78 (Rep78, MW 78 kDa) + his-SUMO illustrating unsuccessful cleavage, molecular weight references for uncleaved proteins shown in respective ‘-SUMO’ lane. White arrows indicate examples of Rep68 (Rep68, MW 68 kDa, example lane 3), and Rep78 (Rep78, MW 78 kDa, example lane 10) after successful cleavage of his-SUMO (MW -18 kDa). Incubation at room temperature (lanes RT) showed incomplete cleavage of his-SUMO.

[0056] Figure 19

[0057] Figure 19 shows genome copy concentrations, measured by qPCR, for chemically defined in vitro capsid filling for AAV5.

[0058] Figure 20

[0059] Figure 20 shows SDS PAGE (left panel) and western blot (right panel) of purified inclusion bodies solubilized with urea. Left panel, lane 1 : PageRuler Plus, lane 2: AAV5 VP3, lane 3: AAV5 VP2, lane 4: AAV5 VP1 . Right panel, lane 1 : AAV5 VP3, lane 2: AAV5 VP2, lane 3: AAV5 VP1 , lane 4. The proteins of interest are indicated with numbered arrows: 1 , AAV5 VP3; 2, AAV5 VP2; 3, AAV5 VP1 .

[0060] Figure 21

[0061] Figure 21 shows SDS PAGE of AAV5 VP3 after ion exchange purification. Lane 1 : PageRuler Plus, lane 2: AAV5 VP3. The protein of interest is indicated with a numbered arrow (1).

[0062] Figure 22

[0063] Figure 22 shows ELISA results of AAV5 VP3 refolding, i.e. , capsid formation, using buffer 1 to 17.

[0064] Figure 23

[0065] Figure 23 shows ELISA results of AAV5 VP3 refolding using buffer 11 (section 7). ON: overnight (16 hours), RT: room temperature (22°C).

[0066] Figure 24

[0067] Figure 24 shows SDS PAGE of AAV5 VP1 , AAV5 VP2, and AAV5 VP3 after ion exchange and gel filtration. Lane 1 : PageRuler Plus, lane 2: AAV5 VP1 , lane 3: AAV5 VP2, lane 4: AAV5 VP3. The proteins of interest are indicated with numbered arrows: 1 , AAV5 VP1 ; 2, AAV5 VP2; 3, AAV5 VP3.

[0068] Figure 25

[0069] Figure 25 shows Western blot of AAV5 capsid particles. Lane 1 : PageRuler Plus, lane 2: 15 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 1 :1 :20; lane 2, 10 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 1 :1 :20; lane 4, 5 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 1 :1 :20; lane 5: 15 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 1 :1 :1 ; lane 6, 10 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 1 :1 :1 ; lane 7, 5 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 1 :1 :1 , lane 8: 15 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 20:20:1 ; lane 9, 10 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 20:20:1 ; lane 10, 5 pL AAV5 VP1 , AAV5 VP2, AAV5 VP3 ratio 20:20:1 . The proteins of interest are indicated with numbered arrows: 1 , AAV5 VP1 ; 2, AAV5 VP2; 3, AAV5 VP3.

[0070] Figure 26

[0071] Figure 26 shows Western blot of purified AAV2-VP3-SUMO produced in P. pastoris before (lane 1), after 1 hour (lane 2), and after 16 hours (lane 3) at room temperature (22°C) in the presence of SUMO and thrombin proteases. The left arrow indicates the protein of interest before cleavage, and the right arrow indicates the protease-cleaved protein of interest.

[0072] Figure 27

[0073] Figure 27 shows ELISA of AAV2 capsids. Samples were measured diluted 10x and 100x in ELISA assay buffer.

[0074] Figure 28

[0075] GFP primer-based qPCR (A) and fill rate result (B). Figure 29

[0076] SDS PAGE of purified MBP-Rep78. Black arrows indicate Rep78 protein (MW 78 kDa) + MBP-linker (MW

[0077] 42.7 kDa).

[0078] Figure 30

[0079] Western blot of purified MBP-Rep78. Black arrows indicate Rep78 protein (MW 78 kDa) + MBP-linker (MW 42.7 kDa). The primary antibody was an anti-AAV2 Replicase mouse mAb, while the secondary was a goat anti-mouse HRP conjugate.

[0080] Figure 31 qPCR (left panel) and fill rate result (right panel).

[0081] Figure 32

[0082] Gaussian fits through the main populations of the chemically defined in vitro filled AAVs (“Fuse”) and HEK-filled AAVs representing the empty, partially filled, and fully filled adeno-associated viruses.

[0083] Definitions

[0084] As used herein, the terms “a” and “an” refer to at least one, i.e., one or more. Accordingly, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0085] As used herein, the term “about” refers to a value that is within 5% above and below the value being described. For example, “pH of about 1 ” as used herein includes quantities that are within 5% above and below pH 1 . Additionally, when used in the context of a list of numerical quantities, it is to be understood that the term “about,” when preceding a list of numerical quantities, applies to each individual quantity recited in the list. For example, “about pH 1 , pH 2, or pH 3” is to be construed as equivalent to individually reciting “about pH 1 ,” “about pH 2,” and “about pH 3.”

[0086] As used herein, the term “adeno-associated virus” (AAV), includes but is not limited to, AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered. See, e.g., Fields et al. Virology, 4th ed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional AAV serotypes and clades have been identified recently. (See, e.g., Gao et al. J. Virol. 78:6381 (2004); Mori et al. Virol. 33:375 (2004). The genomic sequences of various serotypes of AAV, as well as the sequences of the native ITRs, AAV Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature and in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC— 002077, NC— 001401 , NC— 001729, NC— 001863, NC— 001829, NC— 001862, NC— 000883, NC— 001701 , NC— 001510, NC— 006152, NC— 006261 , AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901 , J02275, X01457, AF288061 , AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC— 001358, NC— 001540, AF513851 , AF513852 and AY530579; the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Bantel-Schaal et al. J. Virol. 73:939 (1999); Chiorini et al. J. Virol. 71 :6823 (1997); Chiorini et al. J. Virol. 73:1309 (1999); Gao et al. Proc. Nat. Acad. Sci. USA 99:11854 (2002); Mori et al. Virol. 33:375 (2004); Muramatsu et al. Virol. 221 :208 (1996); Ruffing et al. J. Gen. Virol. 75:3385 (1994); Rutledge et al. J. Virol. 72:309 (1998); Schmidt et al. J. Virol. 82:8911 (2008); Shade et al. J. Virol. 58:921 (1986); Srivastava et al. J. Virol. 45:555 (1983); Xiao et al. J. Virol. 73:3994 (1999); WO 00 / 28061 , WO 99 / 61601 , WO 98 / 11244; and US 6,156,303; the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences.

[0087] As used herein, the term “cell-free” refers to a s a system, a method or a process, such as the assembly of capsids and / or the filling of capsids, in cell free conditions.

[0088] As used herein, the term “in vitro” refers to a system, a method or a process, such as the assembly of capsids and / or the filling of capsids, taking place outside a living organism, such as in a test tube or a culture dish.

[0089] The term “cell-free” and the term “in vitro” might be used interchangeable within the disclosure.

[0090] As used herein, the terms “fill” and “filling,” and the like refer to a series of enzymatically-driven events involving one or more Rep proteins that result in the filling of a capsid with one or more nucleic acid molecules. Thus, when a suitable population of viral capsid proteins (e.g., VP1 , VP2, and / or VP3), conditions, and one or more Rep proteins are combined with a nucleic acid molecule, the respective viral proteins are assembled into a capsid and the capsid can be filled with one or more nucleic acid molecules to form an assembled capsid particle packaged with one or more nucleic acid molecules. The filling process occurs after capsid assembly is complete.

[0091] An “ITR” is a palindromic nucleic acid, e.g., an inverted terminal repeat, that is about 120 nucleotides to about 250 nucleotides in length and capable of forming a hairpin. The term “ITR” includes the site of the viral genome replication that can be recognized and bound by a parvoviral protein (e.g., an AAV Rep protein e.g., Rep78 / 68). An ITR may be from any parvovirus (e.g., an AAV). An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS). The term “ITR” does not require a wild-type parvoviral ITR (e.g., a wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, and missense mutations), as long as the ITR functions to mediate nucleic acid molecule packaging, replication, and / or integration, and the like. The “5’ ITR” is intended to mean the parvoviral ITR located at the 5’ boundary of the nucleic acid molecule; and the term “3’ ITR” is intended to mean the parvoviral ITR located at the 3’ boundary of the nucleic acid molecule.

[0092] “Nucleic acid molecule” and “polynucleotide,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA, which may include naturally occurring and / or synthetically-derived nucleic acids.

[0093] As used herein, the term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter sequence is operably linked to a coding sequence if the promoter sequence promotes transcription of the coding sequence. As another example, an intron sequence is operably linked to a transcriptional unit if the intron contains splice donor and splice acceptor sites allowing for proper splicing of the transcription unit. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two coding regions, contiguous and in reading frame. However, since enhancers may function when separated from the promoter by several kilobases and intronic sequences may be of variable length, some nucleotide sequences may be operably linked but not contiguous.

[0094] The term “parvovirus” as used herein encompasses the family Parvoviridae, including autonomously- replicating parvoviruses and dependoviruses. The autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus, snake parvovirus, and B19 virus. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., Fields et al. Virology, 4th ed. Lippincott-Raven Publishers, Philadelphia, 1996. The genus Dependovirus contains the AAV, including but not limited to, AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, AAV type rh 10, AAV type rh74, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, and ovine AAV.

[0095] “Percent (%) sequence identity” with respect to a reference polynucleotide and polypeptide sequence is defined as the percentage of nucleic acids and amino acids in a coordinate sequence that are identical to the nucleic acids and amino acids in the reference polynucleotide and polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid and amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, and Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid and amino acid sequence, A, to, with, and against a given nucleic acid and amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid and amino acid sequence, A that has a certain percent sequence identity to, with, and against a given nucleic acid and amino acid sequence, B) is calculated as follows:

[0096] 100 multiplied by (the fraction X / Y) where X is the number of nucleotides and amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid and amino acid sequence A is not equal to the length of nucleic acid and amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0097] As used herein, the term “pharmaceutical composition” is intended to also disclose that the capsid included within a pharmaceutical composition can be used for the treatment of the human or animal body by therapy. It is thus meant to be equivalent to the “capsid for use in therapy”.

[0098] As used herein, the term “plasmid” refers to a DNA molecule into which additional DNA segments may be ligated, such as an extrachromosomal circular double-stranded DNA molecule, doggybone DNA (dbDNA), a single-stranded loop, or a capsomere . A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked (e.g., a viral capsid protein and / or a Rep protein).

[0099] As used herein, the term “polynucleotide” refers to a molecule including one or more nucleic acid subunits, and nucleotides, and can be used interchangeably with “nucleic acid” and “oligonucleotide”. A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), and variants thereof. A nucleotide can include a nucleoside and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, and more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five- carbon sugar (either ribose and deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotides and ribonucleic acids, and RNA, can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[0100] As used herein, a “population” refers to a group of at least two (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, or more) proteins of the specified kind (e.g., at least 2 viral capsid proteins or at least 2 VP1).

[0101] As used herein, the term “reaction mixture” refers to a mixture of required components for carrying out a given process, such as the encapsidation or filling of a capsid with a polynucleotide molecule by a capsid in cell free conditions. Such a reaction mixture may entail components such as adenosine triphosphate, a pH buffer, magnesium chloride, radiation, light, and ultraviolent or condition factors such as pH, ionic strength / conductivity, temperature, time, hydrophobicity, space, pressure, and conductivity. Such a reaction mixture may additionally require one or more Rep proteins (e.g., an AAV Rep protein) to enable encapsidation or filling of a capsid with a nucleic acid molecule.

[0102] “Rep proteins” in accordance with the present invention are proteins which fill capsids with a polynucleotide molecule (e.g., viral deoxyribonucleic acid (DNA)) into a capsid. In addition, a Rep protein may enable nucleic acid molecule replication, transcriptional regulation of a nucleic acid molecule, and / or site-specific integration (e.g., chromosomal integration) of a nucleic acid molecule. The Rep protein may be from any parvovirus. As one of skill in the art will appreciate, in some parvovirus family virus species, a Rep protein is referred to as a “non-structural (NS) protein”. As used herein, a Rep protein may refer to a NS protein. As used herein, a Rep protein may refer to an AAV Rep protein, which have been found in all AAV serotypes examined to date, and a synthetic Rep protein. For example, in some embodiments, a AAV Rep protein (e.g., AAV Rep40, AAV Rep52, AAV Rep68, and AAV Rep78) is a capsid protein having an amino acid sequence derived from a particular AAV serotype, for example AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 6.2, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, and ovine AAV. Alternatively, for example, as used herein, a Rep protein may include a synthetic Rep protein. Any suitable Rep protein may be used.

[0103] As used herein, the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek and be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, and be a human and animal who is under care by a trained professional for a particular disease and condition. In preferred embodiments, the subject is a human.

[0104] As used herein, the term “synthetic” may refer to a modified ribonucleotide, a synthetic nucleic acid sequence, or a synthetic protein. A synthetic nucleic acid herein may include an engineered nucleic acid, while a synthetic protein may include an engineered protein (e.g., non-native) and / or a protein including synthetic nucleic or amino acids, such as an engineered protein, nucleic acid, or amino acid not occurring in nature. For example, as used herein, a synthetic protein may include a synthetic viral protein (VP; e.g., VP1 , VP2, and VP3) or a synthetic Rep protein.

[0105] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA) encoding a gene product, such as a peptide, protein, or RNA (e.g., a protein-encoding mRNA or an inhibitory RNA, such as a miRNA, shRNA, or the like). In addition to the coding region for the gene product, the transgene may include, or be operably linked to, one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), destabilizing domain(s), response elements), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Embodiments of the disclosure may utilize any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter elements), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0106] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus, and other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic and eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a gene of interest (e.g., a viral capsid protein, a Rep protein, a nucleic acid molecule, or a reporter protein). Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of a viral capsid protein or a Rep protein as described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of viral capsid proteins or Rep proteins contain polynucleotide sequences that enhance the rate of translation of these genes and improve the stability and nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an IRES, and polyadenylation signal site in and to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, and zeocin.

[0107] As used herein, the term “viral capsid protein” refers to a capsid protein composing a proteinaceous shell. Such a proteinaceous shell is generally composed of one or more viral capsid proteins and when assembled is capable of being loaded with one or more polynucleotide molecules. A viral capsid protein described herein may, for example, be a viral protein (VP) 1 , VP2, and VP3. Further, a viral capsid protein described herein may refer to a synthetic protein or a viral capsid protein from Parvoviridae (e.g., an AAV).

[0108] As used herein, the terms “viral protein 1” and “VP1” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP1 may possess a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (e.g., endocytic entry and receptor-mediated fusion). As used herein, a VP1 may self-assemble into a structure consisting of VP1 , VP2, and / or VP3 molecules. VP1 may exhibit selfbinding properties and self-assemble around the exterior of a respective VP1 -containing capsid. As used herein, a VP1 may be synthetic or a VP1 derived from Parvoviridae (e.g., an AAV). For example, a VP1 derived from an AAV may be a VP1 derived from AAV type 1 , AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered. A VP1 , as used herein, may include an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. Alternatively, and in addition, a VP1 , as used herein, may have an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 2 or 4.

[0109] As used herein, the terms “viral protein 2” and “VP2” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP2 may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell nucleus. As used herein, a VP2 may self-assemble into a structure consisting of VP1 , VP2, and / or VP3 molecules. VP2 may self-assemble within the interior of a respective VP2-containing capsid. As used herein, a VP2 may be synthetic or a VP2 derived from Parvoviridae (e.g., an AAV). For example, a VP2 derived from an AAV may be a VP2 derived from AAV type 1 , AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered. A VP2, as used herein, may include an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. Alternatively, and in addition, a VP2, as used herein, may have an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 13, 16, or 17.

[0110] As used herein, the terms “viral protein 3” and “VP3” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP3 may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell nucleus. As used herein, a VP3 may self-assemble into a structure consisting of VP1 , VP2, and / or VP3 molecules. VP3 may self-assemble within the interior of a respective VP3-containing capsid. As used herein, a VP3 may be synthetic or a VP3 derived from Parvoviridae (e.g., an AAV). For example, a VP3 derived from an AAV may be a VP3 derived from AAV type 1 , AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered. A VP3, as used herein, may include an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. Alternatively, and in addition, a VP3, as used herein, may have an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

[0111] As used herein, the term “P. pastoris” refers to “Pichia pastoris”. As used herein, the term “S. cerevisiae” refers “Saccharomyces cerevisiae”. As used herein, the term “E. coir refers to “Escherichia coli". Detailed Description

[0112] In vitro capsid assembly

[0113] The present inventors have developed a method to enable in vitro AAV capsid assembly. Furthermore, the present inventors have identified several key factors for the in vitro capsid assembly method which can be used to further improve the assembly method. Such in vitro assembly methods have many benefits over conventional methods, including control of viral protein (VP) stoichiometry.

[0114] Method development for sterically controlled in vitro AAV capsid assembly for high capsid titers.

[0115] In some embodiments, the present disclosure relates to a method of in vitro assembling parvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby assembling parvovirus capsids and providing assembled parvovirus capsids.

[0116] Assembly inhibitor

[0117] In some embodiments, the inhibited parvovirus viral capsid proteins are inhibited from assembling by multiple assembly inhibitors, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly inhibitors. In some embodiments, the assembly inhibitor comprises multiple assembly inhibitors, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly inhibitors.

[0118] In some embodiments, the assembly inhibitor is selected from the group consisting of a chemically assembly inhibitor, a steric assembly inhibitor and / or a kinetically assembly inhibitor.

[0119] In some embodiments, the assembly inhibitor is selected from the group consisting of inhibitory pH, inhibitory salt concentration, denaturing, precipitation conditions and / or an antibody.

[0120] In some embodiments, the assembly inhibitor is selected from the group consisting of a retainment of the viral capsid proteins in a compartment and / or physical blockade, such as a cleavable amino acid sequence attached to the viral capsid proteins. In some embodiments, wherein the assembly inhibitor is selected from the group consisting of inhibitory temperature and / or inhibitory viral capsid protein concentration.

[0121] In some embodiments, the assembly inhibitor is selected from the group consisting of an inhibitory modification to the viral capsid proteins, such as a cleavable amino acid sequence, an inhibitory exogenous molecules, inhibitory pH, inhibitory ionic strength, inhibitory conductivity, time, inhibitory temperature, hydrophobicity, inhibiting viral capsid protein concentration, inhibitory viscosity, denaturation, pressure and radiation, such as light, such as ultraviolet light.

[0122] In some embodiments, the assembly inhibitor is an inhibitory modification to the viral capsid proteins. In some embodiments, wherein the assembly inhibitor is a cleavable amino acid sequence attached to the viral capsid proteins. In some embodiments, the cleavable amino acid sequence is a SUMO tag and / or a thrombin cleavage site.

[0123] In some embodiments, the assembly inhibitor is denaturation of the viral capsid proteins.

[0124] In some embodiments the denaturation is thermally-induced denaturation and / or chemically-induced denaturation. In some embodiments, denaturation include a set of conditions which allow the denaturation of parvovirus capsids. In some embodiments, denaturation conditions may include conditions selected from the list including pH, sonication, ionic strength and / or conductivity, or temperature. In some embodiments, denaturation conditions include the factor of pH. In some embodiments, denaturation conditions include sonication. In some embodiments, denaturation conditions include the factor of ionic strength and / or conductivity. In some embodiments, denaturation conditions include the factor of temperature. Denaturation can be performed by any method known in the art.

[0125] In some embodiments, the assembly inhibitor is urea, such as 1 to 15 M urea, 2 to 14 M urea, 3 to 13 M urea, 4 to 12 M urea, 5 to 11 M urea, 6 to 10 M urea or 7 to 9 M urea. In some embodiments, the assembly inhibitor is NaCI, such as 1 to 15 M NaCI, 2 to 14 M NaCI, 3 to 13 M NaCI, 4 to 12 M NaCI, 5 to 11 M NaCI, 6 to 10 M NaCI or 7 to 9 M NaCI. In some embodiments, the assembly inhibitor is KCI, such as 1 to 15 M KCI, 2 to 14 M KCI, 3 to 13 M KCI, 4 to 12 M KCI, 5 to 11 M KCI, 6 to 10 M KCI or 7 to 9 M KCI.

[0126] In some embodiments, the inhibitory pH is pH below or equal to 5, such as pH below or equal to 4.5, such as pH below or equal to 4, such as pH below or equal to 3.5, such as pH below or equal to 3, such as pH below or equal to 2.5, such as pH below or equal to 2, such as pH below or equal to 1 .5. In some embodiments, the inhibitory pH is pH above or equal to 8.5, such as pH above or equal to 9, such as pH above or equal to 9.5, such as pH above or equal to 10, such as pH above or equal to 10.5, such as pH above or equal to 11 , such as pH above or equal to 11 .5, such as pH above or equal to 12, such as pH above or equal to 12.5, such as pH above or equal to 13.

[0127] In some embodiments, the inhibitory temperature is a temperature between -196 °C to 0 °C, such as -190 °C to 0 °C, such as -180 °C to 0 °C, such as -170 °C to 0 °C, such as -160 °C to 0 °C, such as -150 °C to 0 °C, such as -140 °C to 0 °C, such as -130 °C to 0 °C, such as -120 °C to 0 °C, such as -110 °C to 0 °C, such as -100 °C to 0 °C, such as -90 °C to 0 °C, such as -80 °C to 0 °C, such as -70 °C to 0 °C, such as - 60 °C to 0 °C, such as -50 °C to 0 °C, such as -40 °C to 0 °C, such as -30 °C to 0 °C, such as -20 °C to 0 °C, such as -10 °C to 0 °C. In some embodiments, the inhibitory temperature is a temperature between - 196 °C to -1 °C, such as -196 °C to -10 °C, such as -196 °C to -20 °C, such as -196 °C to -40 °C, such as -196 °C to -50 °C, such as -196 °C to -60 °C, such as -196 °C to -70 °C °C, such as -196 °C to -80 °C, such as -196 °C to -90 °C, such as -196 °C to -100 °C, such as -196 °C to -110 °C, such as -196 °C to - 120 °C °C, such as -196 °C to -130 °C, such as -196 °C to -140 °C, such as -196 °C to -150 °C, such as - 196 °C to -160 °C, such as -196 °C to -170 °C, such as -196 °C to -180 °C, such as -196 °C to -190 °C.

[0128] In some embodiments, the inhibitory temperature is a temperature between 40 °C to 280 °C, such as between 40 °C to 250 °C, such as between 40 °C to 220 °C, such as between 40 °C to 210 °C, such as between 40 °C to 200 °C, such as between 40 °C to 190 °C, such as between 40 °C to 180 °C, such as between 40 °C to 170 °C, such as between 40 °C to 160 °C, such as between 50 °C to 160 °C, such as between 60 °C to 160 °C, such as between 70 °C to 160 °C, such as between 80 °C to 160 °C, such as between 80 °C to 150 °C, such as between 80 °C to 140 °C, such as between 90 °C to 140 °C, such as between 90 °C to 140 °C, such as between 100 °C to 140 °C, such as between 100 °C to 130 °C, such as between 110 °C to 130 °C.

[0129] In some embodiments, the inhibitory exogenous molecule is selected from the group consisting of an antibody, a nucleic acid molecule, a peptide, a protein, a nanobody, a lipid, a sugar, a carbohydrate, a polymer, a small molecule, and / or an aptamer.

[0130] In some embodiments, the inhibitory ionic strength and / inhibitory conductivity is a solution comprising NaCI, KCI and / or urea, such as a solution comprising 1 to 15 M NaCI, KCI and / or urea, 2 to 14 M NaCI, KCI and / or urea, 3 to 13 M NaCI, KCI and / or urea, 4 to 12 M NaCI, KCI and / or urea, 5 to 11 M NaCI, KCI and / or urea, 6 to 10 M NaCI, KCI and / or urea or 7 to 9 M NaCI, KCI and / or urea.

[0131] Assembly activator

[0132] In some embodiments, the step of contacting the assembly activator and the inhibited parvovirus viral capsid proteins of step (b) comprise contacting multiple assembly activators, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly activators. In some embodiments, the assembly activator comprises multiple assembly activators, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly activators.

[0133] In some embodiments, the assembly activator is selected from the group consisting of a chemically assembly activator, a sterically assembly activator and / or a kinetically assembly activator.

[0134] In some embodiments, the chemically assembly activator is selected from the group consisting of activating pH, activating salt concentration, renaturing, solubilization conditions and / or removal of the antibody.

[0135] In some embodiments, the sterically assembly activator is selected from the group consisting of a release of the viral capsid proteins from the compartment and / or removal of the physical blockade, such as the cleavable amino acid sequence attached to the viral capsid proteins.

[0136] In some embodiments, the kinetically assembly activator is selected from the group consisting of activating temperature and / or activating viral capsid protein concentration.

[0137] In some embodiments, the assembly activator is selected from the group consisting of a removal of the inhibitory modification to the viral capsid proteins, such as a cleavage of the cleavable amino acid sequence, removal of the inhibitory exogenous molecules, activating pH, activating ionic strength, activating conductivity, activating time, activating temperature, hydrophobicity, activating viral capsid protein concentration, renaturation, activating viscosity, pressure and radiation, such as light, such as ultraviolet light. In some embodiments, the assembly activator is renaturation.

[0138] In some embodiments, the assembly activator is a removal of the inhibitory modification to the viral capsid proteins. In some embodiments, the assembly activator is a cleavage of the cleavable amino acid sequence attached to the viral capsid proteins. In some embodiments, the assembly activator is a protease. In some embodiments, the assembly activator is thrombin and / or a SUMO protease, such as SUMO protease 2. In some embodiments, the cleavable amino acid sequence is a SUMO tag and / or a thrombin cleavage site, and wherein the assembly activator is thrombin and / or a SUMO protease, such as SUMO protease 2.

[0139] In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, such as 1 to 500 mM MES pH 6.0, such as 1 to 450 mM MES pH 6.0, such as 1 to 400 mM MES pH 6.0, such as 1 to 350 mM MES pH 6.0, such as 1 to 300 mM MES pH 6.0, such as 1 to 250 mM MES pH 6.0, such as 1 to 200 mM MES pH 6.0, such as 1 to 150 mM MES pH 6.0, such as 1 to 100 mM MES pH 6.0, such as 1 to 90 mM

[0140] MES pH 6.0, such as 1 to 80 mM MES pH 6.0, such as 1 to 70 mM MES pH 6.0, such as 10 to 70 mM

[0141] MES pH 6.0, such as 20 to 70 mM MES pH 6.0, such as 30 to 70 mM MES pH 6.0, such as 40 to 60 mM

[0142] MES pH 6.0, such as 45 to 55 mM MES pH 6.0.

[0143] In some embodiments, the assembly activator is a buffer comprising NaCI, such as 100 to 1000 mM NaCI, such as 100 to 900 mM NaCI, such as 100 to 800 mM NaCI, such as 100 to 700 mM NaCI, such as 100 to 600 mM NaCI, such as 200 to 600 mM NaCI, such as 300 to 600 mM NaCI, such as 400 to 600 mM NaCI, such as 450 to 550 mM NaCI.

[0144] In some embodiments, the assembly activator is a buffer comprising NaCI, such as 100 to 400 mM NaCI, such as 150 to 400 mM NaCI, such as 200 to 350 mM NaCI, such as 200 to 300 mM NaCI, such as 200 to 290 mM NaCI, such as 210 to 280 mM NaCI, such as 220 to 270 mM NaCI, such as 230 to 240 mM NaCI.

[0145] In some embodiments, the assembly activator is a buffer comprising NaCI, such as 1 to 20 mM NaCI, such as 1 to 15 mM NaCI, such as 5 to 15 mM NaCI, such as 6 to 14 mM NaCI, such as 7 to 13 mM NaCI, such as 6 to 12 mM NaCI, such as 8 to 12 mM NaCI, such as 9 to 11 mM NaCI.

[0146] In some embodiments, the assembly activator is a buffer comprising KCI, such as 1 to 1000 mM KCI, such as 1 to 900 mM KCI, such as 1 to 800 mM KCI, such as 1 to 700 mM KCI, such as 1 to 600 mM KCI, such as 1 to 600 mM KCI, such as 1 to 600 mM KCI, such as 1 to 500 mM KCI, such as 1 to 400 mM KCI, such as 1 to 300 mM KCI, such as 1 to 200 mM KCI, such as 10 to 200 mM KCI, such as 20 to 200 mM

[0147] KCI, such as 30 to 200 mM KCI, such as 40 to 200 mM KCI, such as 50 to 200 mM KCI, such as 50 to

[0148] 150 mM KCI.

[0149] In some embodiments, the assembly activator is a buffer comprising KCI, such as 1 to 20 mM KCI, such as 1 to 15 mM KCI, such as 5 to 15 mM KCI, such as 6 to 14 mM KCI, such as 7 to 13 mM KCI, such as 6 to 12 mM KCI, such as 8 to 12 mM KCI, such as 9 to 11 mM KCI

[0150] In some embodiments, the assembly activator is a buffer comprising KCI, such as 0.1 to 10 mM KCI, 0.1 to 5 mM KCI, 0.1 to 2 mM KCI, 0.1 to 1 mM KCI, 0.1 to 0.5 mM KCI.

[0151] In some embodiments, the assembly activator is a buffer comprising MgCh, such as 0.1 to 20 mM MgCh, such as 0.5 to 20 mM MgCh, such as 1 to 20 mM MgCh, such as 1 to 10 mM MgCh, such as 1 to 9 mM MgCh, such as 1 to 8 mM MgCh, such as 1 to 7 mM MgCh, such as 1 to 6 mM MgCh, such as 1 to 5 mM MgCh, such as 1 to 4 mM MgCh, such as 1 to 3 mM MgCh.

[0152] In some embodiments, the assembly activator is a buffer comprising CaCh, such as 0.1 to 20 mM CaCh, such as 0.5 to 20 mM CaCh, such as 1 to 20 mM CaCh, such as 1 to 10 mM CaCh, such as 1 to 9 mM CaCh, such as 1 to 8 mM CaCh, such as 1 to 7 mM CaCh, such as 1 to 6 mM CaCh, such as 1 to 5 mM CaCh, such as 1 to 4 mM CaCh, such as 1 to 3 mM CaCh.

[0153] In some embodiments, the assembly activator is a buffer comprising Tris-CI, such as 1 to 100 mM Tris-CI, such as 1 to 90 mM Tris-CI, such as 1 to 80 mM Tris-CI, such as 1 to 70 mM Tris-CI, such as 10 to 70 mM Tris-CI, such as 20 to 70 mM Tris-CI, such as 30 to 70 mM Tris-CI, such as 40 to 60 mM Tris-CI.

[0154] In some embodiments, the assembly activator is a buffer comprising Tris-CI, such as 1 to 50 mM Tris-CI, such as 1 to 40 mM Tris-CI, such as 1 to 30 mM Tris-CI, such as 5 to 30 mM Tris-CI, such as 10 to 30 mM Tris-CI, such as 15 to 25 mM Tris-CI.

[0155] In some embodiments, the assembly activator is a buffer comprising Guanidine HCI, such as 0.1 to 5 mM Guanidine HCI, such as 0.1 to 4 mM Guanidine HCI, such as 0.1 to 3 mM Guanidine HCI, such as 0.1 to 2 mM Guanidine HCI, such as 0.5 to 1 .5 mM Guanidine HCI.

[0156] In some embodiments, the assembly activator is a buffer comprising non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, such as 0.01 to 5 % non-ionic surfactant, such as 0.01 to 4 % nonionic surfactant, such as 0.01 to 3 % non-ionic surfactant, such as 0.01 to 2 % non-ionic surfactant, such as 0.1 to 2 % non-ionic surfactant, such as 0.5 to 2 % non-ionic surfactant, such as 0.5 to 1 .5 % non-ionic surfactant, such as 0.5 to 1 % non-ionic surfactant.

[0157] In some embodiments, the assembly activator is a buffer comprising DTT, such as 0.01 to 1 mM DTT, such as 0.01 to 0.5 mM DTT, such as 0.01 to 0.4 mM DTT, such as 0.01 to 0.3 mM DTT, such as 0.01 to 0.2 mM DTT, such as 0.5 to 0.2 mM DTT.

[0158] In some embodiments, the assembly activator is a buffer comprising arginine, such as 0.01 to 10 M arginine, such as 0.01 to 5 M arginine, such as 0.01 to 4 M arginine, such as 0.01 to 3 M arginine, such as 0.01 to 2 M arginine, such as 0.1 to 2 M arginine, such as 0.1 to 1 M arginine, such as 0.2 to 0.9 M arginine, such as 0.3 to 0.8 M arginine, such as 0.4 to 0.7 M arginine, such as 0.4 to 0.6 M arginine.

[0159] In some embodiments, the assembly activator is a buffer comprising polyethylene glycol 3,550, such as 0.001 to 10 % polyethylene glycol 3,550, such as 0.001 to 5 % polyethylene glycol 3,550, such as 0.001 to 1 % polyethylene glycol 3,550, such as 0.001 to 0.5 % polyethylene glycol 3,550, such as 0.01 to 0.5 % polyethylene glycol 3,550, such as 0.01 to 0.1 % polyethylene glycol 3,550.

[0160] In some embodiments, the assembly activator is a buffer comprising GSH, such as 0.01 to 10 mM GSH, such as 0.01 to 5 mM GSH, such as 0.01 to 1 mM GSH, such as 0.01 to 0.5 mM GSH, such as 0.05 to 0.5 mM GSH, such as 0.05 to 0.4 mM GSH, such as 0.05 to 0.3 mM GSH, such as 0.05 to 0.2 mM GSH.

[0161] In some embodiments, the assembly activator is a buffer comprising GSSH, such as 0.001 to 1 mM GSSH, such as 0.001 to 0.5 mM GSSH, such as 0.001 to 0.4 mM GSSH, such as 0.001 to 0.3 mM GSSH, such as 0.001 to 0.2 mM GSSH, such as 0.001 to 0.1 mM GSSH, such as 0.005 to 0.1 mM GSSH, such as 0.005 to 0.05 mM GSSH.

[0162] In some embodiments, the assembly activator is a buffer comprising EDTA, such as 0.1 to 10 mM EDTA, such as 0.1 to 5 mM EDTA, such as 0.1 to 4 mM EDTA, such as 0.5 to 4 mM EDTA, such as 0.5 to 3 mM EDTA, such as 0.5 to 2 mM EDTA.

[0163] In some embodiments, the assembly activator is a buffer comprising sucrose, such as 0.01 to 10 M sucrose, such as 0.01 to 5 M sucrose, such as 0.01 to 2 M sucrose, such as 0.01 to 1 M sucrose, such as 0.1 to 1 M sucrose, such as 0.01 to 0.9 M sucrose, such as 0.01 to 0.8 M sucrose, such as 0.01 to 0.8 M sucrose, such as 0.1 to 0.7 M sucrose, such as 0.1 to 0.6 M sucrose, such as 0.2 to 0.6 M sucrose, such as 0.3 to 0.6 M sucrose, such as 0.3 to 0.5 M sucrose.

[0164] In some embodiments, the assembly activator is a buffer comprising urea, such as 1 to 15 M urea, 2 to 14 M urea, 3 to 13 M urea, 4 to 12 M urea, 5 to 11 M urea, 6 to 10 M urea or 7 to 9 M urea.

[0165] In some embodiments, the assembly activator is a buffer comprising Na2HPO4, such as 1 to 100 mM Na2HPO4, such as 1 to 90 mM Na2HPO4, such as 1 to 80 mM Na2HPO4, such as 1 to 70 mM Na2HPO4, such as 1 to 60 mM Na2HPO4, such as 1 to 50 mM Na2HPO4, such as 1 to 40 mM Na2HPO4, such as 1 to 30 mM Na2HPO4, such as 1 to 20 mM Na2HPO4, such as 5 to 20 mM Na2HPO4, such as 5 to 15 mM Na2HPO4.

[0166] In some embodiments, the assembly activator is a buffer comprising KH2PO4, such as 1 to 100 mM KH2PO4, such as 1 to 90 mM KH2PO4, such as 1 to 80 mM KH2PO4, such as 1 to 70 mM KH2PO4, such as 1 to 60 mM KH2PO4, such as 1 to 50 mM KH2PO4, such as 1 to 40 mM KH2PO4, such as 1 to 30 mM KH2PO4, such as 1 to 20 mM KH2PO4, such as 5 to 20 mM KH2PO4, such as 5 to 15 mM KH2PO4. In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, MgCl2 and / or CaCl2, such as 1 to 100 mM MES pH 6.0, 1 to 20 mM NaCl, 0.01 to 5 mM KCl, 0.1 to 10 mM MgCl2 and / or 0.1 to 10 mM CaCl2, such as 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCl, 0.1 to 1 mM KCl, 0.5 to 4 mM MgCl2 and / or 0.5 to 4 mM CaCl2.In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl and / or KCl, suchas 1 to 100 mM MES pH 6.0, 200 to 300 mM NaCl and / or 1 to 50 mM KCl, such as 30 to 70 mM MES pH 6.0, 220 to 260 mM NaCl and / or 1 to 20 mM KCl, In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, MgCl2,CaCl2, Guanidine HCl, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / orDTT. In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, MgCl2, CaCl2, arginine, polyethylene glycol 3,550, GSH and / or GSSH.In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, EDTA, KCl,sucrose, M Guanidine HCl, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550 and / or DTT.In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, MgCl2,CaCl2, arginine, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, GSH and / or mM GSSH.In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, EDTA,sucrose, Guanidine HCl and / or DTT. In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, EDTA, arginine, sucrose, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550, GSH and / or GSSH. In some embodiments, the assembly activator is a buffer comprising MES pH 6.0, NaCl, KCl, MgCl2, CaCl2, Guanidine HCl, polyethylene glycol 3,550 and / or DTT. In some embodiments, the assembly activator is a buffer comprising mM Tris-Cl pH 8.5, NaCl, KCl, MgCl2, CaCl2, sucrose, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550, GSH and GSSH. In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, NaCl, KCl, EDTA, arginine, Guanidine HCl, polyethylene glycol 3,550 and / or DTT.In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, NaCl, KCl, MgCl2,CaCl2, arginine, sucrose, Guanidine HCl, mM GSH and / or GSSH. In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, mM NaCl, KCl,EDTA, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or DTT.In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, NaCl, KCl, EDTA, polyethylene glycol 3,550, GSH and / or GSSH. In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, NaCl, KCl, 1 mM EDTA, arginine, Guanidine HCl, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or DTT.In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, NaCl, KCl, MgCl2, CaCl2, arginine, sucrose, Guanidine HCl, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 orTriton X-100, polyethylene glycol 3,550, GSH and / or GSSH.In some embodiments, the assembly activator is a buffer comprising Tris-Cl pH 8.5, NaCl, KCl, MgCl2, CaCl2, sucrose and / or DTT. In some embodiments, the assembly activator is a buffer comprising Tris-Cl and / or urea, having pH 8.0. In some embodiments, the assembly activator is a buffer comprising NaCl, KCl, Na2HPO4 and / or 10 mM KH2PO4, having pH 8.0. In some embodiments, the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCl, 0.1 to 1 mM KCl, 1 to 5 mM MgCl2, 1 to 5 mM CaCl2, 0.5 to 1 M Guanidine HCl, 0.1 to 2 %non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or 0.01 to 0.5 mM DTT.In some embodiments, the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCl, 0.1 to 1 mM KCl, 1 to 5 mM MgCl2, 1 to 5 mM CaCl2, 0.05 to 1 M arginine, 0.05 to 0.5 % polyethylene glycol 3,550, 0.01 to 1 m GSSH.In some embodiments, the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 5 to 15mM NaCl, 0.5 to 5 mM EDTA, 0.1 to 1 mM KCl, 0.1 to 1 M sucrose, 0.5 to 1 M Guanidine HCl, 0.05 to 1 % non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.005 to 0.1% polyethylene glycol 3,550 and / or 0.01 to 1 mM DTT.In some embodiments, the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 200 to300 mM NaCl, 1 to 20 mM KCl, 1 to 5 mM MgCl2, 1 to 2 mM CaCl2, 0.05 to 1 M arginine, 0.05 to 1% non- ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.01 to 1 mM GSH and / or 0.001 to 0.1 mM GSSH.In some embodiments, the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 200 to300 mM NaCl, 1 to 20 mM KCl, 0.5 to 5 mM EDTA, 0.1 to 1 M sucrose, 0.5 to 1 M Guanidine HCl and / or 0.01 to 1 mM DTT.In some embodiments, the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 240 mMNaCl, 10 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.4 M sucrose, 0.5% non-ionic surfactant, such asTergitol NP-7, Tergitol NP-10 or Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01mM GSSH.In some embodiments, the assembly activator is a buffer comprising 30 to 70MES pH 6.0, 240 mM NaCl,10 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.75 M Guanidine HCl, 0.05% polyethylene glycol 3,550 and / or 0.1 mM DTT.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 9.6 mM NaCl,0.4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.4 M sucrose, 0.5% non-ionic surfactant, such as Tergitol NP-7,Tergitol NP-10 or Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 9.6 mM NaCl,0.4 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.75 M Guanidine HCl, 0.05% polyethylene glycol 3,550 and / or 0.1 mM DTT.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 9.6 mM NaCl,0.4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.5 M arginine, 0.4 M sucrose, 0.75 M Guanidine HCl, 0.1 mM GSH and / or 0.01 mM GSSH.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 9.6 mM NaCl,0.4 mM KCl, 1 mM EDTA, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X- 100, 0.1 mM DTTIn some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 240 mMNaCl, 10 mM KCl, 1 mM EDTA, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 240 mMNaCl, 10 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.75 M Guanidine HCl, 0.5% non-ionic surfactant, suchas Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or 0.1 mM DTT.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 240 mMNaCl, 10 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.5 M arginine, 0.4 M sucrose, 0.75 M Guanidine HCl, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.In some embodiments, the assembly activator is a buffer comprising 50 mM Tris-Cl pH 8.5, 240 mMNaCl, 10 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.4 M sucrose and / or 0.1 mM DTT.In some embodiments, the assembly activator is a buffer comprising 20 mM Tris-Cl and / or 8 M urea,having pH 8.0. In some embodiments, the assembly activator is a buffer comprising 500 mM NaCl, 100 mM KCl, 10 mM Na2HPO4 and / or 10 mM KH2PO4, having pH 8.0. In some embodiments, the activating time is between 30 minutes and 24 hours, such between 1 hour and 24 hours, such between 1 hour and 20 hours, such between 1 hour and 16 hours. In some embodiments, the activating time is between 15 minutes and 5 hours, such as between 30 minutes and 5 hours, such as between 30 minutes and 4 hours, such as between 30 minutes and 3 hours, such as between 30 minutes and 2 hours, such as between 30 minutes and 1 hour and 30 minutes.In some embodiments, the activating temperature is between 1 ˚C and 45 ˚C, such as between 10 ˚C and45 ˚C, such as between 10 ˚C and 40 ˚C such as between 10 ˚C and 35 ˚C, such as between 10 ˚C and30 ˚C, such as between 10 ˚C and 25 ˚CIn some embodiments, the activating temperature is between 1 ˚C and 30 ˚C, such as between 10 ˚C and 30 ˚C, such as between 10 ˚C and 25 ˚C, such as between 15 ˚C and 25 ˚C, such as between 20 ˚C and 25 ˚C. In some embodiments, the activating temperature is between 15 ˚C and 25 ˚C, such as between 16 ˚C and 25 ˚C, such as between 17 ˚C and 25 ˚C, such as between 17 ˚C and 24 ˚C, such as between 18 ˚C and 24 ˚C, such as between 19 ˚C and 24 ˚C, such as between 20 ˚C and 24 ˚C, such as between 21 ˚C and 24 ˚C, such as between 21 ˚C and 23 ˚C, In some embodiments, the activating temperature is between 1 ˚C and 10 ˚C, such as between 1 ˚C and 9 ˚C, such as between 1 ˚C and 8 ˚C, such as between 1 ˚C and 7 ˚C, such as between 1 ˚C and 6 ˚C, such as between 2 ˚C and 6 ˚C, such as between 3 ˚C and 5 ˚C. Assembly enhancerIn some embodiments the method comprises an assembly enhancer which enhances in vitro assembly.In some embodiments the assembly enhancer is selected from the group consisting of membrane-associated accessory protein, assembly activation protein, an E2A transcriptional regulator, an ICP8 single-stranded DNA-binding protein, presence of any cleavable protein or amino acid sequence, an enzyme that mediates glycosylation, and / or an enzyme that mediates a post translational modification.In some embodiments the assembly enhancer is selected from the group consisting of membrane-associated accessory protein and / or assembly activation protein.In vitro capsid fillingThe present inventors have developed an in vitro parvovirus capsid filling method using scalable andGMP-compatible techniques which enable for clinically relevant and drug development use. Furthermore,the present inventors have identified several key factors for the in vitro capsid filling method which can beused to further improve the filling method. The in vitro filling method also results in reduced DNAtruncation, a significant advantage over current manufacturing technologies as truncation has beenidentified as a major concern by the FDA. The in vitro filling method works for many different AAVserotypes and achieves clinically relevant titers In addition, the filled capsids are more potent.In some embodiments, the present disclosure relates to a method of in vitro filling parvovirus capsids,comprising the steps of: a. providing:i. one or more populations of assembled parvovirus capsids comprising parvovirus viralcapsid proteins in vitro; ii. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain invitro; and iii. a Rep protein in vitro;b. combining the one or more populations of assembled parvovirus capsids, the nucleic acidmolecule, and the Rep protein in vitro under filling conditions which allow filling of theassembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids and providing filled parvovirus capsids.In some embodiments, the present disclosure relates to a method of in vitro filling parvovirus capsidsaccording, comprising the steps of: a. providing:i. a population of assembled parvovirus capsids comprising parvovirus viral capsidproteins in vitro; ii. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain invitro; iii. a Rep proteins in vitro; andiv. a reaction mixture in vitro;b. combining the population of assembled parvovirus capsids, the nucleic acid molecule, theRep protein and the reaction mixture in vitro under filling conditions which allow filling of theassembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids and providing filled parvovirus capsids. Nucleic acid moleculeIn some embodiments, the nucleic acid molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.In some embodiments, the nucleic acid molecule is a viral nucleic acid molecule and / or a non-viral nucleicacid molecule. In some embodiments, the nucleic acid molecule is a viral nucleic acid molecule. In someembodiments, the nucleic acid molecule is a non-viral nucleic acid molecule.In some embodiments, the nucleic aci ucleic acid (DNA) and / orribonucleic acid (RNA). In some embodiments, the nucleic acid molecule comprises DNA. In someembodiments, the nucleic acid molecule comprises RNA. In some embodiments, the nucleic acidmolecule comprises AAV DNA.In some embodiments, the reaction mixture comprises adenosine triphosphate (ATP), guanosinetriphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP),deoxythymidine triphosphate (dTTP), a pH buffer, such as HEPES or MOPS buffer, dithiothreitol, creatinephosphate, creatine phosphokinase (CPK), and / or magnesium chloride. In some embodiments, thereaction mixture comprises adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), a pHbuffer, such as HEPES or MOPS buffer, dithiothreitol, and / or magnesium chloride.Filling conditionsIn some embodiments, the filling conditions are selected from the group consisting of ATP concentration,filling temperature, filling pH, CPK concentration, parvovirus capsid concentration and / or filling time. Insome embodiments, the filling conditions are selected from the group consisting of ATP concentration,filling temperature, filling pH, CPK concentration, an ATP regeneration system, and / or parvovirus capsidconcentration.In some embodiments, the filling conditions comprise ATP concentration. In some embodiments, the ATPconcentration is between 0.1 and 181.44 mM, such as between 0.1 and 180 mM, such as between 0.1and 170 mM, such as between 0.1 and 160 mM, such as between 0.1 and 150 mM, such as between 0.1and 140 mM, such as between 0.1 and 130 mM, such as between 0.1 and 120 mM, such as between 0.1and 110 mM, such as between 0.1 and 100 mM. In some embodiments, the ATP concentration isbetween 0.1 and 100 mM. such as between 1 and 90 mM, such as between 1 and 80 mM, such as between 1 and 70 mM, such as between 1 and 60 mM, such as between 1 and 50 mM, such as between 1 and 40 mM, such as between 1 and 30 mM, such as between 1 and 20 mM, such as between 1 and 15mM, such as between 1 and 10 mM. In some embodiments, the ATP concentration is between 0.01 and10 mM, such as between 0.05 and 10 mM, such as between 0.05 and 9 mM, such as between 0.05 and 8 mM, such as between 0.05 and 7 mM, such as between 0.05 and 6 mM, such as between 0.05 and 5 mM, such as between 0.05 and 4 mM, such as between 0.05 and 3 mM, such as between 0.05 and 2.5mM, such as between 0.1 and 2 mM. In some embodiments, the ATP concentration is between 0.05 and15 mM.In some embodiments, the filling conditions comprise filling temperature. In some embodiments, the fillingtemperature is between 0 °C and 89.7 °C, such as between 5 °C and 80 °C, such as between 5 °C and70 °C, such as between 5 °C and 60 °C, such as between 5 °C and 50 °C, such as between 10 °C and 50°C, such as between 15 °C and 50 °C, such as between 20 °C and 50 °C, such as between 25 °C and 50°C, such as between 30 °C and 50 °C. In some embodiments, the filling temperature is between 30 °Cand 50 °C, such as between 30 °C and 45 °C, such as between 30 °C and 45 °C, such as between 31 °C and 45 °C, such as between 32 °C and 45 °C, such as between 33 °C and 45 °C, such as between 34 °C and 45 °C, such as between 30 °C and 44 °C, such as between 30 °C and 43 °C, such as between 30 °Cand 42 °C, such as between 30 °C and 41 °C. In some embodiments, the filling temperature is between33 °C and 42 °C, such as between 34 °C and 41 °C. In some embodiments, the filling temperature isbetween 35 °C and 40 °C. In some embodiments, the filling temperature is between 1 °C and 40 °C, suchas between 5 °C and 40 °C, such as between 10 °C and 40 °C, such as between 15 °C and 40 °C, such as between 20 °C and 40 °C, such as between 25 °C and 40 °C, such as between 26 °C and 40 °C, suchas between 27 °C and 40 °C, such as between 28 °C and 40 °C, such as between 29 °C and 40 °C, suchas between 29 °C and 39 °C. In some embodiments, the filling temperature is between 20 °C and 50 °C,such as between 20 °C and 45 °C, such as between 20 °C and 40 °C, such as between 25 °C and 40 °C, such as between 26 °C and 40 °C, such as between 27 °C and 40 °C, such as between 28 °C and 40 °C,such as between 29 °C and 40 °C, such as between 29 °C and 39 °C.In some embodiments, the filling conditions comprise filling pH. In some embodiments, the filling pH isbetween 3 and 10, such as between 4 and 9, such as between 4.5 and 9. In some embodiments, thefilling pH is between 5 and 8.8, such as between 5.5 and 8.8, such as between 6 and 8.8, such asbetween 6 and 8.5, such as between 6 and 8. In some embodiments, the filling pH is between 6 and 7.5.In some embodiments, the filling conditions comprise the CPK concentration. In some embodiments, theCPK concentration is between 1 and 1000 ug / ml, such as between 1 and 500 ug / ml, such as between 1 and 400 ug / ml, such as between 1 and 300 ug / ml, such as between 1 and 200 ug / ml, such as between 1and 100 ug / ml. In some embodiments, the CPK concentration is between 1 and 100 ug / ml, such asbetween 1 and 90 ug / ml, such as between 1 and 80 ug / ml, such as between 1 and 70 ug / ml, such as between 1 and 60 ug / ml, such as between 1 and 50 ug / ml, such as between 1 and 40 ug / ml, such asbetween 1 and 30 ug / ml, such as between 1 and 20 ug / ml, such as between 1 and 10 ug / ml. In someembodiments, the CPK concentration is between 5 and 110 ug / ml, such as between 5 and 100 ug / ml,between 5 and 90 ug / ml. In some embodiments, the CPK concentration is between 10 and 100 ug / ml.In some embodiments, the filling conditions comprise an ATP regenerating system. In someembodiments, the ATP regenerating s tyl phosphate as phosphate donor, creatine kinase using creatine phosphate as phosphate donor, pyruvate kinase using phosphoenolpyruvate as phosphate donor, and polyphosphate kinase using polyphosphate as phosphate donor.In some embodiments, the filling conditions comprise the parvovirus capsid concentration. In someembodiments, the parvovirus capsid concentration is between 1e4 and 5 e16 / mL, such as between 1e4and 5 e15 / mL, such as between 1e4 and 1 e15 / mL, such as between 1e4 and 1 e15 / mL, such asbetween 1e4 and 1 e14 / mL, such as between 1e4 and 1 e13 / mL, such as between 1e4 and 1 e12 / mL,such as between 1e4 and 1 e11 / mL, such as between 1e4 and 1 e10 / mL, such as between 1e4 and 1e9 / mL, such as between 1e4 and 1 e8 / mL, such as between 1e4 and 1 e7 / mL, such as between 1e4 and1 e6 / mL, such as between 1e4 and 1 e5 / mL.. In some embodiments, the parvovirus capsid concentrationis between 0.01 and 100 e10 / mL, such as between 0.1 and 100 e10 / mL, such as between 0.1 and 90 e10 / mL. such as between 0.1 and 80 e10 / mL. such as between 0.1 and 70 e10 / mL. such as between 0.1 and 60 e10 / mL. such as between 0.1 and 50 e10 / mL. such as between 0.1 and 40 e10 / mL. such as between 0.1 and 30 e10 / mL. such as between 0.1 and 20 e10 / mL. such as between 0.1 and 10 e10 / mL.such as between 0.1 and 5 e10 / mL. In some embodiments, the parvovirus capsid concentration isbetween 0.01 and 10 e10 / mL, such as between 0.1 and 10 e10 / mL, such as between 0.5 and 10 e10 / mL, such as between 0.5 and 9 e10 / mL, such as between 0.5 and 8 e10 / mL, such as between 0.5 and 7 e10 / mL, such as between 0.5 and 6 e10 / mL, such as between 0.5 and 5 e10 / mL. In some embodiments, the parvovirus capsid concentration is between 0.5 and 7 e10 / mL.In some embodiments, the reaction mixture comprises HEPES, such as 30mM HEPES, MES buffer,phosphate buffer, Tris buffer or MOPS buffer. In some embodiments, the filling conditions comprisesHEPES, such as 30mM HEPES, MES buffer, phosphate buffer, Tris buffer or MOPS buffer.In some embodiments, the filling conditions comprises the following:a. the ATP concentration is between 0.05 and 15 mM;b. the filling temperature is between 35 °C and 40 °C;c. the CPK concentration is between 10 and 100 ug / ml; and / ord. the parvovirus capsid concentration is between 0.5 and 7 e10 / mL.In some embodiments, the filling conditions comprises filling time. In some embodiments, the filling time isbetween 0.5 and 24 hours, such as between 0.5 and 20 hours, such as between 0.5 and 16 hours, such as between 0.5 and 12 hours, such as between 0.5 and 10 hours, such as between 1 and 10 hours, such as between 1 and 9 hours, such as between 2 and 8 hours, such as between 2 and 7 hours, such as between 3 and 6 hours, such as betwe diments, the filling time is between 1 and 7 hours, such as between 2 and 7 hours, such as between 2.5 and 6.5 hours, such as between 3and 6 hours, such as between 3 and 5 hours, such as between 3.5 and 4.5 hours. In some embodiments,the filling time is between 3 and 5 hours.In some embodiments, the filling conditions comprises the following:a. the ATP concentration is between 0.05 and 15 mM;b. the filling temperature is between 35 °C and 40 °C;c. the CPK concentration is between 10 and 100 ug / ml;d. the parvovirus capsid concentration is between 0.5 and 7 e10 / mL; and / ore. the filling time is between 3 and 5 hours.In some embodiments, the nucleic acid molecule comprises a first inverted terminal repeat (ITR1) and / ora second inverted terminal repeat (ITR2). In some embodiments, the ITR1 and / or the ITR2 is from AAV.In some embodiments, the ITR1 and / or the ITR2 comprises the Rep binding domain. In someembodiments, the nucleic acid molecule, the ITR1 and / or the ITR2 are operably linked to each other in a5’-to-3’ direction. In some embodiments, the nucleic acid molecule, the ITR1 and / or the ITR2 are operablylinked to each other in a 3’-to-5’ directionIn some embodiments, the method comprises a filling enhancer which enhances in vitro filling. fillingenhancer which enhances in vitro assembly. In some embodiments, the filling enhancer is selected fromthe group consisting of membrane-associated accessory protein, assembly activation protein, an E2A transcriptional regulator, an ICP8 single-stranded DNA-binding protein, presence of any cleavable protein or amino acid sequence, an enzyme that mediates glycosylation, and / or an enzyme that mediates a posttranslational modification. In some embodiments, the filling enhancer is selected from the groupconsisting of membrane-associated accessory protein and / or assembly activation protein. In someembodiments, the E2A transcriptional regulator is from adenovirus. In some embodiments, the ICP8single-stranded DNA-binding protein is from herpes virus. In some embodiments, the enzyme thatmediates a post translational modification is synthetic.In some embodiments, the filling conditions are conditions which denature the assembled capsids. Insome embodiments, the filling conditions which denature are condition factors selected from this list comprising: pH, sonication, ionic strength / conductivity, or temperature. In some embodiments, the filled parvovirus capsids comprises an increased amount of intact nucleic acid molecule compared to parvovirus capsids that have been filled using conventional methods. In someembodiments, the filled parvovirus cap eic acid molecule, such as an 85% intact nucleic acid molecule, such as a 90% intact nucleic acid molecule, such as a 95% intact nucleic acid molecule, such as a 96% intact nucleic acid molecule, such as a 97% intact nucleic acid molecule, such as a 98% intact nucleic acid molecule, such as a 99% intact nucleic acid molecule, suchas a 100% intact nucleic acid molecule. In some embodiments, the filled parvovirus capsids comprise a100% intact nucleic acid molecule.In some embodiments, the nucleic acid molecule comprised in the filled parvovirus capsids has reducedtruncation compared to a nucleic acid molecule comprised in parvovirus capsid that have been filled usingconventional methods. In some embodiments, the nucleic acid molecule comprised in the filled parvoviruscapsids has 20% truncation, such as 15% truncation, such as 10% truncation, such as 5% truncation, such as 4% truncation, such as 3% truncation, such as 2% truncation, such as 1% truncation, such as 0%truncation. In some embodiments, the nucleic acid comprises the ITR1, the transgene of interest and theITR2.In some embodiments, at least 80% of the assembled parvovirus capsids are filled by said method, suchas at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or wherein at least 99% ofthe assembled parvovirus capsids are filled by said method. In some embodiments, 100% of theassembled parvovirus capsids are filled by said method. In some embodiments, at least 80% of the assembled parvovirus capsids are filled with said transgene of interest by said method, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or wherein at least 99% of the assembled parvovirus capsids are filled by said method. In some embodiments, 100% of the assembled parvovirus capsids are filled with said transgene of interest by said method. In some embodiments, the method yields at least 80% filled parvovirus capsids, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97% least 99% filled parvoviruscapsids. In some embodiments, the method yields 100% filled parvovirus capsids.In vitro manufacturing of filled capsidsThe present inventors have developed an in vitro method of manufacturing complete assembled and filledparvovirus capsids. Furthermore, the present inventors have identified several key factors for the in vitrocapsid assembly method and the in vitro capsid filling method which can be used to further improve the invitro manufacturing method.In some embodiments, the present disclosure relates to a method of in vitro manufacturing filledparvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro,wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; andc. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b)in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby providing one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro; d. providing:i. a nucleic acid molecule comprising a transgene of interest and a Rep bindingdomain in vitro; and ii. a Rep protein in vitro;e. combining the population of assembled parvovirus capsids obtained in c., the nucleic acidmolecule and the Rep protein in vitro under filling conditions which allow filling of theassembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids, thereby manufacturing filled parvovirus capsids.In some embodiments, the present disclosure relates to a method of in vitro manufacturing filledparvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro,wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited pa ro; andc. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b)in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby providing one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro d. providing:i. a nucleic acid molecule comprising a transgene of interest and a Rep binding domainin vitro; ii. a Rep protein in vitro; andiii. a reaction mixture in vitroe. combining the population of assembled parvovirus capsids obtained in c., the nucleic acidmolecule, the Rep protein and the reaction mixture in vitro under filling conditions which allowfilling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids, thereby manufacturing filled parvovirus capsids.In some embodiments, the assembly step and / or the filling step is as defined elsewhere in the presentdisclosure. Viral capsid proteins In some embodiments, the viral capsid proteins are from a Densovirinae, Hamaparvovirinae, or Parvovirinae.In some embodiments, the viral capsid proteins are from a Parvovirinae selected from the groupconsisting of an Amdoparvovirus, Artiparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetraparvovirus, preferably Dependoparvovirus.In some embodiments, the viral capsid proteins are from an adeno-associated virus (AAV).In some embodiments, the viral capsid proteins are synthetic viral capsid proteins and / or recombinantviral capsid proteins. In some embodiments, the viral capsid protein is a synthetic protein (e.g., an engineered protein and / or a protein composed of synthetic nucleic some embodiments, a synthetic capsid may include the synthetic capsids described in WO 2011 / 054976, WO 2019 / 158619, and WO 2020 / 068990, incorporated herein in their entirety by reference. A synthetic viral capsid protein may be any suitable synthetic viral capsid protein now known in the art or later discovered.In some embodiments, the viral capsid proteins are selected from the group consisting of viral protein 1(VP1), viral protein 2 (VP2), and / or viral protein (VP3). In some embodiments, the viral capsid proteins areVP1. In some embodiments, the viral capsid proteins are VP2. In some embodiments, the viral capsidproteins are VP3. In some embodiments, the viral capsid proteins are VP1 and VP2. In someembodiments, the viral capsid proteins are VP1 and VP3. In some embodiments, the viral capsid proteinsare VP2 and VP3. In some embodiments, the viral capsid proteins are VP1, VP2, and VP3. In someembodiments, the VP1, VP2 and / or VP3 are AAV VP1, AAV VP2 and / or AAV VP3.In some embodiments, the AAV is an AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAVtype 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, rh10 AAV, rh.39 AAV, or any other AAV now known and later discovered.In some embodiments, the VP1, VP2 and / or VP3 are synthetic VP1, VP2 and / or VP3 and / or recombinantVP1, VP2 and / or VP3. In some embodiments, the VP is a VP from an AAV type 1. In some embodiments, the VP is a VP from an AAV type 2. In some embodiments, the VP is a VP from an AAV type 3. In some embodiments, the VPis a VP from an AAV type 4. In some embodiments, the VP is a VP from an AAV type 5. In someembodiments, the VP is a VP from an AAV type 6. In some embodiments, the VP is a VP from an AAV type 7. In some embodiments, the VP is a VP from an AAV type 8. In some embodiments, the VP is a VP from an AAV type 9. In some embodiments, the VP is a VP from an AAV type 10. In some embodiments, the VP is a VP from an AAV type 11. In some embodiments, the VP is a VP from an AAV type 12. In some embodiments, the VP is a VP from an AAV type 13. In some embodiments, the VP is a VP from an avian AAV. In some embodiments, the VP is a VP from a bovine AAV. In some embodiments, the VP is a VP from a canine AAV. In some embodiments, the VP is a VP from a goat AAV. In some embodiments, the VP is a VP from a snake AAV. In some embodiments, the VP is a VP from an equine AAV. In some embodiments, the VP is a VP from an ovine AAV. In some embodiments, the VP is a VP from a goat AAV. In some embodiments, the VP is a VP from a shrimp AAV. In some embodiments, the VP is a VP from a rh10 AAV. In some embodiments, the VP is a VP from a rh74 AAV.In some embodiments, the VP1 is a VP1 from AAV serotype 1, serotype 2, serotype 3A, serotype 3B,serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10,serotype 11, serotype 12, serotype 13, serotype rh10, or serotype rh74. In some embodiments, the VP2is a VP2 from AAV serotype 1, serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13,serotype rh10, or serotype rh74. In some embodiments, the VP3 is a VP3 from AAV serotype 1, serotype2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, serotype rh10, or serotype rh74.In some embodiments, the VP1, VP2, and / or VP3 are from different AAV serotypes. In someembodiments, the VP1, VP2, and / or VP3 are from the same AAV serotype.In some embodiments, the viral capsid proteins, such as VP1, VP2 and / or VP3, is produced in aneukaryotic microbial system, such as yeast, such as a methylotrophic yeast. In some embodiments, theviral capsid proteins, such as VP1, VP2 and / or VP3, is produced in an eukaryotic microbial system, such as P. pastoris or S. cerevisiae. In some embodiments, the viral capsid proteins, such as VP1, VP2 and / orVP3, is produced in an prokaryotic microbial system, such as E. Coli. In some embodiments, the viralcapsid proteins, such as VP1, VP2 and / or VP3, is produced a mammalian cell system, such as HEK, CHO, HeLa or Vero cells, an insect cell system, such as Sf9, Sf21 or High Five cells, a plant system, suchas tobacco BY, an algae system, a metazoan cell system, a zebrafish and / or cell-free expression systems. In some embodiments, the viral capsid proteins further comprise a purification tag, such as a His-tag, a C- tag (EPEA), a streptavidin tag and / or biotin tag. In some embodiments, the viral capsid protein is a viral capsid protein exemplified by the Parvoviridae viral proteins in Table 1, below: Table 1: Exemplary wild-type amino acid sequences of Parvoviridae viral proteins

[0167]

[0168] In some embodiments, the VP1 is a VP1 from AAV serotype 1. In some embodiments, the VP1 is a VP1 from AAV serotype 2. In some embodiments, the VP1 is a VP1 from AAV serotype 3. In some embodiments, the VP1 is a VP1 from AAV serotype 4 In some embodiments, the VP1 is a VP1 from AAV serotype 5. In some embodiments, the VP1 is a VP1 from AAV serotype 6. In some embodiments, the VP1 is a VP1 from AAV serotype 7. In some embodiments, the VP1 is a VP1 from AAV serotype 8. In some embodiments, the VP1 is a VP1 from AAV serotype 9. In some embodiments, the VP1 is a VP1from AAV serotype 10. In some embodiments, the VP1 is a VP1 from AAV serotype 11. In some embodiments, the VP1 is a VP1 from AAV serotype 12. In some embodiments, the VP1 is a VP1 from AAV serotype 13. In some embodiments, the VP1 is a VP1 from AAV serotype rh10. In some embodiments, the VP1 is a VP1 from AAV serotype rh74. In some embodiments, the VP1 is a VP1 exemplified by the AAV VP1 in Table 2 and Table 3, below:Table 2: Exemplary wild-type amino acid sequences of AAV VP1

[0169] In some embodiments, the VP1 is a VP1 from AAVrh74, for example, as described in WO 2015 / 013313, WO 2006 / 110689, WO 2013 / 123503, or WO 2013 / 158879, incorporated herein in their entirety by reference. In some embodiments, the VP2 is a VP2 from AAV serotype 1. In some embodiments, the VP2 is a VP2 from AAV serotype 2. In some embodiments, the VP2 is a VP2 from AAV serotype 3. In some embodiments, the VP2 is a VP2 from AAV serotype 4. In some embodiments, the VP2 is a VP2 fromAAV serotype 5. In some embodiments, the VP2 is a VP2 from AAV serotype 6. In some embodiments, the VP2 is a VP2 from AAV serotype 7. In some embodiments, the VP2 is a VP2 from AAV serotype 8. In some embodiments, the VP2 is a VP2 from AAV serotype 9. In some embodiments, the VP2 is a VP2 from AAV serotype 10. In some embodiments, the VP2 is a VP2 from AAV serotype 11. In some embodiments, the VP2 is a VP2 from AAV serotype 12. In some embodiments, the VP2 is a VP2 from AAV serotype 13. In some embodiments, the VP2 is a VP2 from AAV serotype rh10. In some embodiments, the VP2 is a VP2 from AAV serotype rh74. In some embodiments, the VP2 is a VP2 exemplified by the AAV VP2 in Table 4, below: In some embodiments, the VP3 is a VP3 from AAV serotype 1 . In some embodiments, the VP3 is a VP3 from AAV serotype 2. In some embodiments, the VP3 is a VP3 from AAV serotype 3. In some embodiments, the VP3 is a VP3 from AAV serotype 4. In some embodiments, the VP3 is a VP3 from AAV serotype 5. In some embodiments, the VP3 is a VP3 from AAV serotype 6. In some embodiments, the VP3 is a VP3 from AAV serotype 7. In some embodiments, the VP3 is a VP3 from AAV serotype 8. In some embodiments, the VP3 is a VP3 from AAV serotype 9. In some embodiments, the VP3 is a VP3 from AAV serotype 10. In some embodiments, the VP3 is a VP3 from AAV serotype 11 . In some embodiments, the VP3 is a VP3 from AAV serotype 12. In some embodiments, the VP3 is a VP3 from AAV serotype 13. In some embodiments, the VP3 is a VP3 from AAV serotype rh 10. In some embodiments, the VP3 is a VP3 from AAV serotype rh74.

[0170] In some embodiments, the VP3 is a VP3 exemplified by the AAV VP3 in Table 5, below:

[0171] Table 5: Exemplary wild-type amino acid sequences of AAV VP3

[0172]

[0173] In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 86% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 87% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 88% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 89% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 91 % identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 92% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 93% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 94% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 96% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 97% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 98% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10. In some embodiments, the VP1 comprises or consists of an amino acid sequence that is identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7- 10.

[0174] In some embodiments, the VP1 has an amino acid sequence that is at least 85% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10.

[0175] In some embodiments, the VP1 is inhibited from assembling by an assembly inhibitor.

[0176] In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 85% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 86% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 87% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 88% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 89% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 91 % identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 92% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 93% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 94% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 96% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 97% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 98% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17. In some embodiments, the VP2 comprises or consists of an amino acid sequence that is identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

[0177] In some embodiments, the VP2 has an amino acid sequence that is at least 85% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

[0178] In some embodiments, the VP2 is inhibited from assembling by an assembly inhibitor.

[0179] In some embodiments, the VP2 is a VP2 from AAV serotype 2, such as, for example, a VP2 with UniProt ID No.: P03135-2 (SEQ ID NO: 5).

[0180] In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 85% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 86% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 87% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 88% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 89% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 91 % identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 92% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 93% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 94% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 96% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 97% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 98% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24. In some embodiments, the VP3 comprises or consists of an amino acid sequence that is identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

[0181] In some embodiments, the VP3 has an amino acid sequence that is at least 85% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

[0182] In some embodiments, the VP3 is inhibited from assembling by an assembly inhibitor.

[0183] Ratio of viral capsid proteins

[0184] In some embodiments, the one or more populations of inhibited parvovirus viral capsid proteins and / or the one or more populations of assembled parvovirus capsids comprise a specific ratio between VP1 , VP2 and / or VP3. In some embodiments, the one or more populations of inhibited parvovirus viral capsid proteins and / or the one or more populations of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60.

[0185] In some embodiments, the assembled parvovirus capsids and / or filled parvovirus capsids comprise a specific ratio between VP1 , VP2 and / or VP3. In some embodiments, the assembled parvovirus capsids and / or filled parvovirus capsids parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60.

[0186] Further method steps

[0187] Purification

[0188] In some embodiments, the methods disclosed here further comprises a step of purifying, a step of formulating and / or a step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids.

[0189] The assembled parvovirus capsids and / or the filled parvovirus capsids disclosed herein may be purified by any suitable purification method known in the art, such as, for example, chromatography, ultracentrifugation, centrifugation, flocculation, filtration, and / or ultrafiltration / diafiltration.

[0190] In some embodiments, the method further comprises a step of purifying the assembled parvovirus capsids and / or the filled parvovirus capsids. In some embodiments, the step of purifying comprises chromatography, ultra-centrifugation, centrifugation, flocculation, filtration, and / or ultrafiltration / diafiltration. In some embodiments, the capsids are purified by chromatography. In some embodiments, the capsids are purified by ultra-centrifugation. In some embodiments, the capsids are purified by centrifugation. In some embodiments, the capsids are purified by flocculation. In some embodiments, the capsids are purified by filtration. In some embodiments, the capsids are purified by ultrafiltration / diafiltration

[0191] Formulation

[0192] Mixtures of the capsids described herein may be prepared in water suitably mixed with one or more excipients, carriers, and diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0193] In some embodiments, the method further comprises a step of formulating the assembled parvovirus capsids and / or the filled parvovirus capsids into a formulation, optionally wherein the assembled parvovirus capsids and / or the filled parvovirus capsids are purified assembled parvovirus capsids and / or purified filled parvovirus capsids. In some embodiments, the step of formulating comprises formulating the assembled parvovirus capsids and / or the filled parvovirus capsids into a formulation with trehalose, sugar, sucrose, polyols, and / or dimethylsulfoxide. In some embodiments, the step of formulating comprises formulating the assembled parvovirus capsids and / or the filled parvovirus capsids into a formulation with a pharmaceutically acceptable carrier, diluent and / or excipient. In some embodiments, the pharmaceutically acceptable carrier, diluent and / or excipient is selected from the group consisting of tween 20, tween 80, trehalose, Pluronic F68, sorbitol and / or glycerol, such as 0.0001 to 0.01 % Pluronic F68, 1 to 10 % sorbitol and / or 1 to 10% glycerol.

[0194] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions and dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions and dispersions. In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent and dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars and sodium chloride.

[0195] Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0196] Furthermore, the capsids described herein can be formulated into pharmaceutical compositions for administration to a patient in a biologically compatible form suitable for administration in vivo. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.

[0197] A pharmaceutical composition containing, for example, a capsid described herein would typically include a pharmaceutically acceptable diluent and carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and a nucleic acid. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and a nucleic acid. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and a nucleic acid. The sterile PBS is typically a pharmaceutical grade PBS.

[0198] In certain embodiments, pharmaceutical compositions include one or more capsids and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

[0199] In certain embodiments, capsids may be admixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions and formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease to be treated, and dose to be administered.

[0200] In certain embodiments, pharmaceutical compositions including a capsid encompass any pharmaceutically acceptable salts of the inhibitor, esters of the inhibitor, and salts of such esters. In certain embodiments, pharmaceutical compositions including a capsid, upon administration to a subject (e.g., a human), are capable of providing (directly and indirectly) the polynucleotide molecule (e.g., payload). Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of inhibitors, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0201] Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs include one or more conjugate group attached to a capsid, wherein the conjugate group is cleaved by endogenous nucleases within the body.

[0202] In certain embodiments, pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

[0203] In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues and cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

[0204] In certain embodiments, pharmaceutical compositions include a co-solvent system. Certain of such cosolvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant Polysorbate 80™ and 65% w / v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars and polysaccharides may substitute for dextrose.

[0205] In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, etc.). In certain of such embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water and physiologically compatible buffers such as Hanks's solution, Ringer's solution, and physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility and serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules and in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions, and emulsions in oily and aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate and triglycerides, and liposomes.

[0206] Storage

[0207] The assembled parvovirus capsids and / or the filled parvovirus capsids disclosed herein may be stored under conditions that maintain capsid integrity. For example, storage conditions may include temperature, state of the capsid (e.g., solid, aerosolized, lyophilized), storage reagents, pH, and pressure.

[0208] In some embodiments, the method further comprises a step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids, optionally wherein the storing is under conditions that maintain capsid integrity. In some embodiments, the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids is at room temperature. In some embodiments, the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids is at 1 °C to 25 °C, such as 1 °C to 20 °C, such as 5 °C to 25 °C, such as 10 °C to 25 °C, such as 15 °C to 25 °C. In some embodiments, the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids comprises lyophilizing and aerosolizing the assembled parvovirus capsids and / or the filled parvovirus capsids. In some embodiments, the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids at a low pH or a high pH. In some embodiments, the method comprises a step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids under pressure.

[0209] Rep Proteins

[0210] Filling of the capsids described herein may include filling with one or more Rep proteins, which may be a synthetic Rep protein or a naturally occurring Rep protein. In some embodiments, the synthetic Rep protein is an engineered protein and / or a protein composed of synthetic amino acids.

[0211] In some embodiments, Rep protein is a recombinant Rep protein and / or a recombinantly produced Rep protein.

[0212] In some embodiments, the Rep protein is a purified Rep protein. In some embodiments, the Rep protein is purified from Escherichia Coli. In some embodiments, the Rep protein comprises multiple Rep proteins, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 Rep proteins. In some embodiments, the step of providing a Rep protein is a step of providing multiple Rep proteins, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 Rep proteins

[0213] In some embodiments, the Rep protein is a synthetic Rep protein. In some embodiments, the Rep protein is produced in an eukaryotic microbial system, such as yeast, such as a methylotrophic yeast. In some embodiments, the Rep protein is produced in an eukaryotic microbial system, such as P. pastoris or S. cerevisiae. In some embodiments, the Rep protein is produced in an prokaryotic microbial system, such as E. Coli. In some embodiments, the Rep protein is produced in a mammalian cell system, such as HEK, CHO, HeLa or Vero cells, an insect cell system, such as Sf9, Sf21 or High Five cells, a plant system, such as tobacco BY, an algae system, a metazoan cell system, a zebrafish and / or cell-free expression systems

[0214] In some embodiments, the one or more Rep proteins is a Rep protein of Parvoviridae (e.g., Densovirinae, Hamaparvovirinae, or Parvovirinae). In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Densovirinae, Hamaparvovirinae, or Parvovirinae. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Densovirinae. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Hamaparvovirinae.

[0215] In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Parvovirinae (e.g., a Amdo parvovirus, Artiparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetraparvovirus). In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Amdoparvovirus, Artiparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetra parvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Amdoparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Artiparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Aveparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Bocaparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Copiparvoviru. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Dependoparvovirus (e.g., an AAV). In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Erythroparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Loriparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Protoparvovirus. In some embodiments, the one or more Rep proteins is a Rep protein is a Rep protein of Tetraparvovirus. In some embodiments, the one or more Rep proteins may include Rep40, Rep52, Rep68, and Rep78. In some embodiments, the one or more Rep proteins may include AAV Rep40, AAV Rep52, AAV Rep68, and AAV Rep78.

[0216] In some embodiments, one or more Rep proteins is a Rep protein from an AAV. In some embodiments, the AAV is an AAV type 1 , AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, rh 10 AAV, rh.39 AAV, or any other AAV now known and later discovered. For example, in some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 1 . In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 2. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 3. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 4. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 5. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 6. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 7. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 8. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 9. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 10. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 11. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 12. In some embodiments, the one or more Rep proteins is a Rep protein from an AAV type 13. In some embodiments, the one or more Rep proteins is a Rep protein from an avian AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a bovine AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a canine AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a goat AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a snake AAV. In some embodiments, the one or more Rep proteins is a Rep protein from an equine AAV. In some embodiments, the one or more Rep proteins is a Rep protein from an ovine AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a goat AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a shrimp AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a rh 10 AAV. In some embodiments, the one or more Rep proteins is a Rep protein from a rh74 AAV. In some embodiments, the one or more Rep proteins is a Rep protein from an any other AAV now known and later discovered.

[0217] In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74. For example, in some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 1 . In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 2. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 3. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 4. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 5. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 6. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 7. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 8. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 9. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 10. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 11. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 12. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype 13. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype rh 10. In some embodiments, the one or more Rep proteins is a Rep protein from AAV serotype rh74.

[0218] Any suitable combination of one or more Rep proteins may be used in the methods described herein. For example, in some embodiments, the one or more Rep proteins is Rep40. In some embodiments, the one or more Rep proteins is Rep52. In some embodiments, the one or more Rep proteins is Rep40 and Rep52. In some embodiments, the one or more Rep proteins is Rep68. In some embodiments, the one or more Rep proteins is Rep78. In some embodiments, the one or more Rep proteins is Rep68 and Rep78. In some embodiments, the one or more Rep proteins is Rep40, Rep52, Rep68, and Rep78. In some embodiments, the one or more Rep proteins is Rep40, Rep52, and Rep68. In some embodiments, the one or more Rep proteins is Rep40, Rep52, and Rep78.

[0219] In some embodiments, the one or more Rep proteins is AAV Rep40. In some embodiments, the one or more Rep proteins is AAV Rep52. In some embodiments, the one or more Rep proteins is AAV Rep40 and AAV Rep52. In some embodiments, the one or more Rep proteins is AAV Rep68. In some embodiments, the one or more Rep proteins is AAV Rep78. In some embodiments, the one or more Rep proteins is AAV Rep68 and AAV Rep78. In some embodiments, the one or more Rep proteins is AAV Rep40, AAV Rep52, AAV Rep68, and AAV Rep78. In some embodiments, the one or more Rep proteins is AAV Rep40, AAV Rep52, and AAV Rep68. In some embodiments, the one or more Rep proteins is AAV Rep40, AAV Rep52, and AAV Rep78.

[0220] In some embodiments, the Rep40, Rep52, Rep68, and / or Rep78 are from different AAV serotypes. In some embodiments, the Rep40, Rep52, Rep68, and / or Rep78 are from the same AAV serotypes. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 35, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 36, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 37, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 38, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 39, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 40, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 41 , optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 41 . In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 42, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 43, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43.

[0221] In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 35. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 36. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 37. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 38. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 39. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 40. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 41 . In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 42. In some embodiments, the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 43.

[0222] For example, in some embodiments, the Rep protein is a Rep protein exemplified by the AAV Rep proteins in Table 6, below:

[0223] Table 6: Exemplary AAV Rep Proteins

[0224] In some embodiments, the Rep protein concentration is between 0.01 and 100 pM, such as between 0.01 and 10 pM, between 0.01 and 5 pM, between 0.01 and 2.5 pM, between 0.01 and 1 pM, between 0.01 and 0.5 pM, between 0.01 and 0.25 pM, between 0.01 and 0.1 pM, between 0.1 and 100 pM, between 0.1 and 10 pM, between 0.1 and 5 pM, between 0.1 and 2.5 pM, between 0.1 and 1 pM, between 0.1 and 0.5 pM, between 0.1 and 0.25 pM, between 0.25 and 100 pM, between 0.25 and 10 pM, between 0.25 and 5 pM, between 0.25 and 2.5 pM, between 0.25 and 1 pM, between 0.25 and 0.5 pM, between 0.5 and 100 pM, between 0.5 and 10 pM, between 0.5 and 5 pM, between 0.5 and 2.5 pM, between 0.5 and 1 pM, between 1 and 100 pM, between 1 and 10 pM, between 1 and 5 pM, between 1 and 2.5 pM, between 2.5 and 100 pM, between 2.5 and 10 pM, between 2.5 and 5 pM, between 5 and 100 pM, between 5 and 10 pM, or such as wherein the Rep protein concentration is between 10 and 100 pM. In some embodiments, the Rep protein concentration is 0.1 to 10 pM, such as 0.1 to 9 pM, such as 0.1 to 8 pM, such as 0.1 to 7 pM, such as 0.1 to 6 pM, such as 0.1 to 5 pM, such as 0.5 to 5 pM, such as 0.5 to 4 pM, such as 0.5 to 3 pM, such as 0.5 to 2 pM, such as 0.5 to 1 .5 pM. In some embodiments, the Rep protein concentration is 1 pM.

[0225] ITR

[0226] In some embodiments, the ITR1 and / or the ITR2 are from a Parvoviridae. In some embodiments, the ITR1 and / or the ITR2 are from a Densovirinae, Hamaparvovirinae, or Parvovirinae. In some embodiments, the ITR1 and / or the ITR2 are from a Amdoparvovirus, Artiparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetraparvovirus. In some embodiments, the ITR1 and / or the ITR2 are from a Dependoparvovirus. In some embodiments, the ITR1 and / or the ITR2 are from an AAV. In some embodiments, the ITR1 and / or the ITR2 are from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74. In some embodiments, the ITR1 and / or the ITR2 are from AAV serotype 2. In some embodiments, the VP1 , VP2 and / or VP3, the Rep protein, and / or ITR1 and / or ITR2 are from different AAV serotypes. In some embodiments, the VP1 , VP2 and / or VP3, the Rep protein, and / or ITR1 and / or ITR2 are from the same AAV serotype.

[0227] AAV Rep fusion proteins

[0228] In some embodiments, the present disclosure relates to an AAV Rep fusion protein comprising: a. An AAV Rep domain or a AAV Rep protein, and b. A cleavable amino acid sequence, such as a SUMO tag.

[0229] In some embodiments, the AAV Rep protein is selected from the group consisting of Rep40, Rep52, Rep68 and / or Rep78. In some embodiments, the AAV Rep fusion protein further comprises a purification tag, such as a His-tag, a C-tag (EPEA), a streptavidin tag and / or biotin tag.

[0230] In some embodiments, the AAV Rep protein is as defined elsewhere in the present disclosure.

[0231] In some embodiments, the present disclosure relates to a fusion protein, such as a MBP-Rep78 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 35, such as 81%, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 35.

[0232] In some embodiments, the present disclosure relates to a fusion protein, such as a MBP-Rep78 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 35.

[0233] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep40 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 36, such as 81%, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 36.

[0234] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep40 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 36.

[0235] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep52 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 37, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 37.

[0236] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep52 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 37.

[0237] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep68 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 38, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0238] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep68 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 38.

[0239] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep78 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 39, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 39.

[0240] In some embodiments, the present disclosure relates to a fusion protein, such as a SUMO-Rep78 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep40 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 40, such as 81%, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0241] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep40 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 40.

[0242] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep52 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 41 , such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 41 .

[0243] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep52 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 41.

[0244] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep68 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 42, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0245] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep68 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 42.

[0246] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep78 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 43, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 43.

[0247] In some embodiments, the present disclosure relates to a fusion protein, such as a Smt3-Rep78 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 43.

[0248] Vectors encoding AAV Rep fusion proteins and method of producing AAV Rep fusion proteins

[0249] In some embodiments, the present disclosure relates to a vector encoding the AAV Rep fusion protein according to the present disclosure.

[0250] In some embodiments, the present disclosure relates a method of producing the AAV Rep fusion protein according to the present disclosure s, comprising the steps of: a. Transforming a prokaryotic microbial system, such as E. coli, with the vector encoding the AAV Rep fusion protein according to the present disclosure; and b. Incubating the prokaryotic microbial system, such as E. coli, thereby producing the AAV Rep fusion protein.

[0251] In some embodiments, the present disclosure relates a method of producing the AAV Rep fusion protein according to the present disclosure, comprising the steps of: a. Transforming an eukaryotic microbial system, such as yeast, such as P. pastoris or S. cerevisiae, with the vector encoding the AAV Rep fusion protein according to the present disclosure, b. Incubating the eukaryotic microbial system, such as P. pastoris, or S. cerevisiae, thereby producing the AAV Rep fusion protein.

[0252] In some embodiments, the method further comprises a step of inoculating the prokaryotic microbial system or the eukaryotic microbial system. In some embodiments, the method further comprises a step of purifying the fusion protein. In some embodiments, the eukaryotic microbial system is a methylotrophic yeast.

[0253] AAV VP fusion protein

[0254] In some embodiments, the present disclosure relates an AAV VP fusion protein comprising: a. An AAV VP domain or an AAV VP protein, and b. A cleavable amino acid sequence, such as a SUMO tag.

[0255] In some embodiments, the AAV VP protein is selected from the group consisting of AAV VP1 , AAV VP2, and / or AAV VP. In some embodiments, the AAV VP fusion protein further comprises a purification tag, such as a His-tag, a C-tag (EPEA), a streptavidin tag and / or biotin tag.

[0256] In some embodiments, the AAV VP protein comprises: a. A purification tag, such as a His-tag, b. A cleavable amino acid sequence, such as a SUMO tag, c. An AAV VP protein, such as AAV VP1 , AAV VP2 and / or AAV VP3, d. A thrombin cleavage site, e. A cleavable amino acid sequence, such as a SUMO tag, and f. A purification tag, such as C-tag (EPEA).

[0257] In some embodiments, the AAV VP protein is as defined elsewhere in the present disclosure.

[0258] Vectors encoding AAV VP fusion proteins and method of producing AAV VP fusion proteins

[0259] In some embodiments, the present disclosure relates to a vector encoding the AAV VP fusion protein according to the present disclosure.

[0260] In some embodiments, the present disclosure relates to a method of producing a method of producing the AAV VP fusion protein according to the present disclosure, comprising the steps of: a. Transforming a prokaryotic microbial system, such as E. coli, with the vector encoding the AAV VP fusion protein according to the present disclosure; and b. Incubating the prokaryotic microbial system, such as E. coli, thereby producing the AAV VP fusion protein.

[0261] In some embodiments, the present disclosure relates to a method of producing the AAV fusion protein according to the present disclosure, comprising the steps of: a. Transforming a eukaryotic microbial system, such as yeast, such as P. pastoris or S. cerevisiae, with the vector encoding the AAV VP fusion protein according to the present disclosure b. Incubating the eukaryotic microbial system, such as P. pastoris or S. cerevisiae, thereby producing the AAV fusion protein.

[0262] In some embodiments, the method further comprises a step of inoculating the prokaryotic microbial system or the eukaryotic microbial system. In some embodiments, the method further comprises a step of purifying the fusion protein. In some embodiments, the eukaryotic microbial system is a methylotrophic yeast.

[0263] Kit of parts

[0264] In some embodiments, the present disclosure relates to a kit of parts comprising: a. one or more populations of inhibited parvovirus viral capsid proteins of the present disclosure; b. an assembly inhibitor of the present disclosure; c. an assembly activator of the present disclosure; and d. optionally instructions for use.

[0265] In some embodiments, the present disclosure relates to a kit of parts comprising: a. one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins of the present disclosure; b. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain of the present disclosure; c. a Rep protein of the present disclosure; and d. optionally instructions for use.

[0266] In some embodiments, the present disclosure relates to a kit of parts comprising: a. one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins of the present disclosure; b. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain of the present disclosure; c. a Rep protein of the present disclosure; d. a reaction mixture of the present disclosure; and e. optionally instructions for use.

[0267] In some embodiments, the present disclosure relates to a kit of parts comprising: a. one or more populations of inhibited parvovirus viral capsid proteins of the present disclosure; b. an assembly inhibitor of the present disclosure; c. an assembly activator of the present disclosure; d. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain of the present disclosure; e. a Rep protein of the present disclosure; and f. optionally instructions for use. In some embodiments, the present disclosure relates to a kit of parts comprising: a. one or more populations of inhibited parvovirus viral capsid proteins of the present disclosure; b. an assembly inhibitor of the present disclosure; c. an assembly activator of the present disclosure; d. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain of the present disclosure; e. a Rep protein of the present disclosure; f. a reaction mixture of the present disclosure; and g. optionally instructions for use.

[0268] Population of assembled or filled capsids

[0269] In some embodiments, the present disclosure relates to a population of assembled parvovirus capsids obtainable by the method according to the present disclosure. In some embodiments, the population of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60. In some embodiments, the population of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in a ratio of 1 :1 :10 (VP1 :VP2:VP3).

[0270] In some embodiments, the present disclosure relates to a population of filled parvovirus capsids obtainable by the method according to the present disclosure. In some embodiments, the population of filled parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60. In some embodiments, the population of filled parvovirus capsids comprises VP1 , VP2 and VP3 in a ratio of 1 :1 :10 (VP1 :VP2:VP3).

[0271] Alternative methods for in vitro capsid filling

[0272] In some embodiments, the present disclosure relates to a method of in vitro filling parvovirus capsids, comprising: a) providing: i) a population of assembled parvovirus capsids comprising one or more parvovirus viral proteins; and ii) a nucleic acid molecule; and b) combining the population of assembled parvovirus capsids and the nucleic acid molecule comprising the nucleic acid molecule under conditions which allow the encapsidation of the nucleic acid molecule into the assembled capsids, wherein at least 1 % of the population of assembled capsids comprise capsids encapsidating one or more nucleic acid molecules, thereby manufacturing capsids.

[0273] In some embodiments, the present disclosure relates to a method of in vitro filling a method of manufacturing capsids, comprising:

[0274] (a) providing:

[0275] (i) a population of assembled parvovirus capsids comprising parvovirus viral capsid proteins;

[0276] (ii) a set of conditions which allow the denaturation of the assembled parvovirus capsids; and

[0277] (iii) a nucleic acid molecule (e.g., containing a transgene of interest); and

[0278] (b) combining the population of assembled parvovirus capsids and the nucleic acid molecule comprising the nucleic acid molecule under conditions which denature the assembled capsids; and

[0279] (c) contacting an activator and the denatured capsids comprising parvovirus capsid proteins, wherein the activator allows the re-assembly of capsids comprising parvovirus capsid proteins into assembled capsids and allows the encapsidation of the nucleic acid molecule into the assembled capsids, thereby manufacturing capsids.

[0280] In some embodiments, step

[0281] (a) further comprises providing

[0282] (iv) one or more Rep proteins, and wherein step

[0283] (c) further comprises contacting an activator and the denatured capsids, wherein the activator allows the re-assembly of capsids comprising parvovirus capsid proteins into assembled capsids and allows filling of the assembled capsids with the nucleic acid molecule by the one or more Rep proteins.

[0284] In some embodiments the denaturation is thermally-induced denaturation and / or chemically-induced denaturation. In some embodiments, denaturation include a set of conditions which allow the denaturation of parvovirus capsids. In some embodiments, denaturation conditions may include conditions selected from the list including pH, sonication, ionic strength and / or conductivity, or temperature. In some embodiments, denaturation conditions include the factor of pH. In some embodiments, denaturation conditions include sonication. In some embodiments, denaturation conditions include the factor of ionic strength and / or conductivity. In some embodiments, denaturation conditions include the factor of temperature. Denaturation can be performed by any method known in the art. Examples

[0285] The following examples are put forth so as to provide those of and ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0286] Example 1 : Scalable, lysate based capsid filling Introduction

[0287] Adeno-Associated Virus (AAV) is the most widely used viral vector for gene therapy and the leading viral vector for direct in-vivo clinical applications (Wang, 2019). AAV consists of two major components: A protein capsid, and a single-stranded DNA genome. In a clinical setting, the viral genome can be replaced by an Inverted Terminal Repeat (ITR) flanked Gene of Interest (Gol). AAV first assembles an icosahedral procapsid, genome packaging proteins, Rep protein, bind to the capsid creating a portal a the 5-fold vertex. Rep proteins demonstrate helicase and nuclease activity, translocating single-stranded DNA into the capsid, in an ATP- dependent fashion. A major challenge in AAV drug development is achieving a high proportion of vectors that are packaged with DNA - also referred to as filled capsids. In industrial processes, most viral capsids will often not contain genomic material, and filled capsid yields are highly variable (Heldt, et al., 2023). It has been postulated that this could be due to the method these vectors are produced: In cell-based expression systems. Such systems are (1) inherently complex; (2) highly variable in the sense that minor process variations can have an amplified effect on drug product quality; (3) not solely geared to the production of a therapeutic agent, rendering such platforms highly inefficient for production of viral vectors.

[0288] Based on the above, the present inventors have hypothesized that producing AAV in an in vitro (i.e., cell- free) system can produce a high proportion of filled capsids in a consistent and predictable manner. Zhou et al (Zhou, 1998) conducted a study to understand the mechanisms of AAV propagation by AAV capsid filling using a cell lysate-based system.. The work described by Zhou et al is not an ideal capsid filling process and is not implementable for drug development or clinical applications due to the following limitations:

[0289] • Requirement of adherent cells, rendering the method poorly scalable.

[0290] • Requirement of medium containing serum, rendering the method incompatible with GMP-grade production

[0291] • Requirement of medium containing antibiotics, rendering the method incompatible with GMP- grade production • Requirement of pre-infection with Adenovirus to enable AAV protein expression, rendering the method less compatible with GMP-grade production

[0292] • Requirement of ultra-gradient centrifugation, rendering the method poorly scalable and incompatible with GMP-grade production. • Lack of analytical resolution for process monitoring or making quantitative conclusions about product quality.

[0293] The present invention addresses the above limitations and provides an improved in vitro (i.e. , cell free) method of filling capsids. The present invention relates to a scalable, clinically relevant cell-free ( / n vitro) AAV capsid filling method as shown in Figure 1 . The in vitro filling method of the present invention has the following advantages:

[0294] • Increased process scalability

[0295] • GMP compatibility

[0296] • Increased capsid filling yield.

[0297] • Use of quantitative assays (ELISA and qPCR) for process monitoring and assessing filling efficiency.

[0298] Equipment and materials

[0299] Equipment

[0300] Materials

[0301] Methods

[0302] Preparative procedures

[0303] Component generation

[0304] Plasmid generation

[0305] Three plasmids were purchased from Cell Biolabs:

[0306] • pAAV-RC2 (cat no.: VPK-402): Containing the AAV2 Rep gene and the AAV2 Cap gene. The annotated sequence is listed in ’’Sequence Overview” (SEQ ID NO: 25). • pHelper Vector (cat no.: VPK-422): Containing the Adenovirus E2A gene; the Adenovirus E4 gene; the Adenovirus VA gene. The annotated sequence is listed in ’’Sequence Overview” (SEQ ID NO: 26).

[0307] • pAAV-GFP Control Vector (cat no.: AAV-400): Containing GFP reporter gene; a CMV promotor region; a Poly A sequence; and two flanking ITR regions. The annotated sequence is listed in ’’Sequence Overview” (SEQ ID NO: 27). E. coli transformations for all vectors were performed by Synvolux BV (Leiden, The Netherlands). pAAV- RC2 and pHelper were transformed into NEB-DH5a cells; pAAV-GFP was transformed into NEB-stable cells(data not shown).

[0308] Plasmids were amplified through fermentation as follows:

[0309] • +- 10pL of glycerol stock was used to inoculate LB medium with 0.1 mg / mL ampicillin in baffled Nalgene shake flasks. For pAAV-RC2, this was done using a 1 L flask with 400mL medium; for pAAV-GFP and pHelper this was done with 2 x 500mL with 200mL medium.

[0310] • Fermentation was performed at 30°C, 180 rpm for 20 hours.

[0311] • For plasmid purification, a Maxi Prep procedure (ThermoFisher Scientific, 2011) was followed, with the following observations and deviations: o Pellets post-harvest centrifugation for pAAV-GFP and pHelper were significantly larger than for the other plasmids. This is likely due to the culture being overgrown, thus having more biomass. o Due to a lack of resuspension buffer (R3), 10mL of dPBS was added to 30mL of R3 to achieve the minimum total resuspension volume. An excess of RNAase (1 OOpL) was added to this 10mL dPBS fraction. o Lysis was carried out for 7 minutes, instead of 5 minutes. o Plasmid were resuspended in 500pL of TE buffer.

[0312] • Plasmid yields were as follows: o pAAV-RC2: 1086.1 ng / pL o pAAV-RC5: 2826.3 ng / pL o pAAV-GFP: 446.2 ng / pL o pHelper: 747.9 ng / pL

[0313] • Plasmids were diluted in TE buffer to a target concentration of 0.25mg / mL (250ng / pL).

[0314] Cell lysate generation

[0315] VPC 2.0 cells were expanded for five passages in Viral Production Medium with GlutaMax. Incubation was at 8% CO2, 37°C, 85% RH, and 120rpm (10mm orbital diameter). On day 2 of the fifth passage, the Viable Cell Density (VCD) was 4.75 x 106VC / mL; with a viability of 98.7%. This was diluted in prewarmed medium to a concentration of 3 x 106VC / mL. For transfection the AAV-MAX Helper-Free AAV Production System guide was used (Gibco, 2023), with the following notes: • Culture volume: 240mL (in 2L unbaffled vented shake flask)

[0316] • VCD: 3 x 106VC / mL

[0317] • AAV Max enhancer: 2400pL

[0318] • Viral plex complexation buffer volume: 24mL

[0319] • Transfection booster: 720pL

[0320] • Transfection reagent: 1440pL

[0321] • pHelper: 556pL

[0322] • pAAV-RC2: 884 pL

[0323] • Complexation incubation time: 25 minutes

[0324] After 70 hours, VCD was 4.45 x 106VC / mL and the culture viability was 86.5%. The culture was then split into two 125mL cultures in 1 L shake flasks. The first flask was treated with 12.5mL AAV Max lysis buffer; the second flasks was treated with 12.5mL AAV Max lysis buffer, 250pL 1 M MgCh, and 500pL of Pierce nuclease solution (@25kll / mL).

[0325] After a two-hour incubation at 37°C and 120rpm, lysates were centrifuged in 50mL Greiner tubes at 4500xg, 30 minutes, 4°C.

[0326] Glycerol was added to each aliquot to achieve 20% v / v concentration. Material was stored at -20±5°C.

[0327] Capsid concentration was determined as described under “Capsid quantification”. Note, Rep concentration was not determined.

[0328] Upon use for this work, one 55mL aliquot (lacking nuclease) was thawed and distributed in 2mL aliquots. One of these aliquots was required for this experiment. The remaining aliquots were stored at -20°C.

[0329] Capsid AAV Packaging Matrix preparation

[0330] Stock solution preparation

[0331] The following (stock) solutions were prepared:

[0332] • HEPES 30mM, pH adjusted with NaOH and HCI to 7.5, 0.2pm sterile filtered, stored at RT.

[0333] • DTT 0.87M in milliQ water. 0.2pm sterile filtered, stored at -20+-5°C.

[0334] • Creatine Phosphate (CP) disodium salt tetrahydrate 0.2M; in aforementioned HEPES buffer. 0.2pm sterile filtered, stored at -20+-5°C.

[0335] • Creatine Phosphokinase (CPK) from rabbit muscle, Type 1 (1 mg / mL; 310u / mL); Merck -C3755- 3.5KU; Source SLCK 9545; Cas No.: 9001-15-4; in aforementioned HEPES buffer. 0.2pm sterile filtered, stored at -20+-5°C. Inactive CAPM preparation

[0336] 20mL inactive CAPM (lacking enzymes) medium was prepared as described in Table 7.

[0337] Table 7: Inactive CAPM components

[0338] Inactive CAPM was not filtered and stored in 1 .925 mL aliquots at -20+-5°C.

[0339] In vitro capsid filling

[0340] To activate CAPM medium, 75pL CPK solution was added to 1 .925 mL inactive CAPM. Six experimental conditions were performed (Table 8):

[0341] Table 8: Cell free capsid filling conditions

[0342] Analytics

[0343] Capsid quantification

[0344] Progen’s AAV2 Xpress ELISA kit protocol was used to determine capsid concentration (Progen, 2023), with the following notes:

[0345] • Measurement wavelength: 450nm

[0346] • Reference wavelength: 620nm

[0347] • Measurement performed on a Tecan Genios Pro plate reader.

[0348] • Duplicate samples

[0349] • Undiluted samples and 100-fold diluted samples were measured.

[0350] Results are shown in Figure 2.

[0351] Genome copy quantification

[0352] Takara’s AAVpro® Titration Kit (for Real Time PCR) Ver.2 kit protocol was used to determine genome copy concentration (Takara, 2023), with the following notes:

[0353] • During DNA extraction (protocol step VI-2) extravirion DNA impurities are digested using DNAse, before capsid lysis.

[0354] • To test for extravirion DNA digestion efficiency using DNAse two samples of pure pGFP were added (250pg / mL) as negative controls: one to be measured directly without DNAse digestion, and one digested as per the protocol.

[0355] • Measurement was performed on a ThermoFisher Scientific QuantStudio5 Real-Time PCR System. • For each sample a 10-, 100- , 1000- and 10000-fold diluted samples were measured.

[0356] Results are shown in Figures 3-5.

[0357] Conclusions

[0358] In summary, the present inventors have developed a method using scalable and GMP-compatible techniques to enable in vitro AAV capsid filling for clinically relevant and drug development use.

[0359] Furthermore, several observations can be made from the generated data:

[0360] Figure 2 shows a standard curve used to determine total capsid concentration, which was found to be 1 .86e10 capsids / mL for the material used in capsid filling experiments. Adjusting for experimental dilution, this was fixed at fixed at 8.6e9 capsid / mL. This value is almost 1000X higher than the estimate by Zhou et al (1 e7 capsid / mL). This demonstrates that the developed method is much more suitable for generating material for drug development and clinical purposes.

[0361] Figure 3 and Figure 5 show that increasing plasmid additions results in higher genome copy levels when capsid concentration is kept constant, demonstrating that plasmid concentration can be used as a parameter to control in vitro capsid filling.

[0362] Figure 3 also indicates that the achieved filling titers are significantly higher (>1000X) than suggested by Zhou et al - 1 .37e8 gc / mL versus an approximated amount of 1 e5 infectious particles / mL. This was further confirmed in Example 3 by providing a direct comparison with a cell-based assay.

[0363] The trend in Figure 4 shows that filled capsid are being detected with qPCR as opposed to an artifact generated during qPCR preparations by undigested unencapsulated plasmid. This was further confirmed in Example 3 by addition of a purification (and free-DNA removal) step.

[0364] The plasmid packing efficiency trend in Figure 5 indicates that an increase in added plasmid results in a decrease in plasmid packaging efficiency. This implies that plasmid concentration is not the only driver of in vitro capsid filling. As such, Example 2 investigated additional parameters aiming to further improve the described method.

[0365] Example 2: Capsid filling improvement

[0366] Introduction

[0367] Example 1 demonstrated a method using scalable and GMP-compatible techniques to enable in vitro AAV capsid filling for clinically relevant and drug development use.

[0368] The in vitro method of Example 1 outperformed methods described by Zhou et al, and the highest observed filling ratio was 1 .6% of all capsids. Industry standards typically range from 5-80% and the present inventors decided to further improve the method of Example 1 . Furthermore, Example 1 indicated that plasmid packaging was somehow being limited by yet unidentified bottlenecks. The present inventors sought to improve in vitro capsid filling reactions by performing a half-fractional 2-level factorial screening experiment for the following factors and ranges: • pH: 6; 6.7; 7.5

[0369] • Temperature (°C): 35; 37; 39

[0370] • [ATP] (mM): 0.1 ; 1.05; 2

[0371] • [CPK] (pg / mL): 10; 55; 100

[0372] • Empty capsid concentration at start of reaction (e10 / mL): 1 , 3, 5

[0373] Equipment and materials

[0374] Equipment

[0375] Materials

[0376] Methods

[0377] Experimental design

[0378] The following experimental design was generated use JMP 17 software (Table 9):

[0379] Table 9 Screening design

[0380] Preparative procedures

[0381] Buffer preparation

[0382] Three 10mL solutions were prepared: • HEPES 30mM, pH 7.5

[0383] • HEPES 30mM, pH 6.75

[0384] • HEPES 30mM, pH 6.0

[0385] To each, 327 mg Creatine Phosphate disodium tetrahydrate (CP) was added to achieve a final concentration of 0.1 M. This was finally supplemented with the following (Table 10):

[0386] Table 10 Base buffer components

[0387] The following working solutions were used:

[0388] • CPK solution (1000|jg / mL): prepared as described in Example 1 .

[0389] • Clarified lysate - containing rep and empty capsids: same material as used in Example 1 .

[0390] • GFP plasmid (0.41 pg / pL): 25pL purified plasmid at 4.1 pg / mL with 225pL HEPES 30mM, pH 6.75

[0391] • Empty capsid spiking solution (6.8e11 capsids / mL): 100pL 6.8e12 empty AAV2 capsids / mL (Progen) with 900pL HEPES 30mM, pH 6.75

[0392] • ATP solution (10mM): 50pL 100mM solution (Thermo) with 450pL HEPES 30mM, pH 6.75

[0393] In vitro capsid filling

[0394] Three round deep-well plates were used: one for each temperature condition. These were placed on ice prior to loading reagents. Wells were filled with components as described in Table 11 and in the following sequence:

[0395] 1 . ATP solution was added, as per design

[0396] 2. Capsid spiking solution was added, as per design

[0397] 3. 10pL plasmid working stock was added to each well

[0398] 4. 245pL Cell lysate from Example 1 was added to each well

[0399] 5. To initiate the reaction, CPK was added last, as per design

[0400] Table 11 Design space, where appropriate, converted to volumetric additions (pL) of working solutions.

[0401] Deep well plates were sealed, placed in Thermomixers with temperature setpoints as specified in Table 11 and incubated at 750rpm for four hours. Analytics qPCR analysis was performed as described in Example 1 . Any results below the limit of quantification were considered 0. Results are shown in Figure 6 and corresponding conditions are summarized in Table 12 below.

[0402] Table 12 qPCR results from capsid filling condition screening

[0403] Model generation, Analysis of Variance, and local optimum prediction qPCR results were used as the response in this analysis and were imported into the above design and analyzed using JMP 17.

[0404] A main effects and interactions model was generated. This was done by selecting all possible first-power terms of the investigated factors for stepwise regression and generating all possible models. The model taken forward for analysis was selected based on having the lowest RMSE. Model terms and corresponding significance are listed in Table 13. The relative contribution of an individual factor was defined as the total of the sum of squares of each factor-associated term as a percentage of the total of the sum of squares of all terms in the screening model (Table 14).

[0405] Table 13 Model terms and significance: Statistically significant (p<0.05) model terms are highlighted with “*”.

[0406] Table 14 Relative factor contribution to model variance Conclusion

[0407] Through DoE-based screening, within the investigated design space, the present inventors have demonstrated that [ATP], pH, T, [CPK] and [Capsids] significantly drive in vitro capsid filling (Table 13, Table 14). In addition, the present inventors have demonstrated that in vitro capsid filling is heavily dependent on multiple factor interactions (Table 13). Finally, the present inventors have identified several conditions that significantly improve titers of in vitro capsid filling, with a maximum observed titer of 2.39e10 gc / mL (Figure 6, Table 12). This represents a 174.4X increase in genome copy titer compared to results from Example 1 .

[0408] Example 3: Verification and purification

[0409] Introduction

[0410] Example 1 demonstrated that the present inventors had developed a scalable and GMP-compatible techniques to enable in vitro AAV capsid filling for clinically relevant and drug development use. Example 2 demonstrated that the in vitro capsid filling is driven by [ATP], pH, T and [CPK].

[0411] Based on findings from Example 2, the present inventors selected the following conditions to maximize material generation:

[0412] • [ATP]=10mM

[0413] • T=39°C

[0414] • pH=6

[0415] • [CPK]=10pg / mL

[0416] • [Capsid]=1e10 / mL

[0417] The experiment was scaled up, this served several purposes:

[0418] 1 . Demonstrated the in vitro filling unit operation is scalable.

[0419] 2. Benchmark in vitro filled AAV quality against AAV produced using a scalable manufacturing process. The present inventors expanded the suite QC tests to include potency analysis through a cell based GFP reporter assay.

[0420] 3. Using in vitro filling, produce sufficient material to facilitate a post-filling purification. This maximizes host cell DNA removal, providing the highest degree of confidence in qPCR titers reflecting filled capsid.

[0421] This experimental overview is shown in Figure 7. Equipment and materials

[0422] Equipment

[0423] Materials Additional materials described here cover infectivity analysis, all other materials have been previously described in Example 1 and Example 2

[0424] Methods

[0425] Preparative procedures Buffer preparation

[0426] 20mL inactive CAPM (lacking enzymes) medium was prepared as described in Table 15.

[0427] Table15: Inactive CAPM components

[0428] The following working solutions were used:

[0429] • CPK solution (1000|jg / mL): prepared as described in Example 1

[0430] • Clarified lysate - containing rep and empty capsids: same material as used in Example 1 • GFP plasmid (0.41 pg / pL): 10OpL purified plasmid at 4.1 pg / mL with 225pL HEPES 30mM, pH

[0431] 6.75

[0432] • ATP solution (10mM): 50pL 100mM solution (Thermo) with 450pL HEPES 30mM, pH 6.75

[0433] In vitro filling of capsids Filling was performed by mixing empty AAV2 capsids, cell lysate containing AAV2 Rep proteins, a ITR CMV-GFP ITR containing plasmid, and a Capsid AAV Packaging Matrix (CAMP) together with creatine phosphokinase (CPK) (see Table 15 for CAMP composition and Table 16 for final in vitro packaging setup). As controls, in vitro filling experiments without either AAV2 empty capsids, or plasmid, or both components were used (Table 16).

[0434] Table 16. In vitro filling setup

[0435] The filling was performed by incubation at 39°C for 4 hours and material was subsequently purified as described in ”AAV purification”. AA V purification

[0436] Chromatography was performed using an AKTA Pure, with online measurements of pH, conductivity, and UV absorbance (260 and 280 nm). The 1 ml AAVX adsorbent column was equilibrated and AAV material loaded and eluted as below (Table 17). Upon elution the AAV material was diluted in 1 M Tris, pH 9.0 until a neutral pH (pH 7.4) was achieved.

[0437] Table 17.Chromatography phases for AAVX chromatographic purification of AAV. | | I | | | I l 1 | I |j

[0438] Samples were stored at -80°C until analyzed.

[0439] Control AAV2-GFP vector

[0440] AAV2-GFP control vectors were produced in a standard transient triple transfection system. Briefly, VPC 2.0 Hek293 cells (ThermoFisher Scientific) were transfected with pRC2, pHelper, and pAAV-GFP using AAVX-Max transfection reagent (ThermoFisher Scientific). After three days expression, the cells were harvested and lysed. The obtained lysate was clarified by centrifugation and 0.22 pm filtration, and subsequently purified on a AAVX (ThermoFisher Scientific) chromatography column. The vectors were stored at -80°C until analyzed. Analytics

[0441] Capsid quantification

[0442] Capsid quantification analysis was performed by ELISA as described in Example 1 (results are shown in Figure 10).

[0443] Genome copy quantification qPCR analysis was performed as described in Example 1 . Any results below the limit of quantification were considered 0 (results shown in Figure 9).

[0444] Cell based infectivity analysis

[0445] HEK293 cells were cultured in an incubator at 37°C in a 5% (v / v) CO2 enriched atmosphere at 95% humidity, supplemented with 10% (v / v) FBS, 1 % (v / v) penicillin / streptomycin. To test infectivity of the in vitro filled AAV vectors, human HEK293 cells were seeded into a 96-well cell culture plate at 15000 cells / well. The day after, the cells were transduced with the in vitro filled AAV vectors and control AAV vectors by adding the vectors directly to medium at the following titers: 5 VG / cell, 50 VG / cell, and 500 VG / cell.

[0446] 48 hours after the transduction, the cells were analyzed by fluorescence microscope and flow cytometry (Attune Flow Cytometer) for GFP expression. For flow analysis, the gating strategy is shown in Figure 8 (results shown in Figure 12).

[0447] Conclusions

[0448] Through a direct comparison of in vitro filled AAV material, and AAV material generated using a conventional triple transfection method (control AAV2) the present inventors show that:

[0449] 1 . A comparable qPCR titer was observed for the in vitro filled AAV2 when comparing to control AAV2 (Figure 9).

[0450] 2. The percentage of filled capsids is higher for in vitro filled AAV2 (11 % filled capsids) compared to control AAV2 (6% filled capsids) (Figure 10B).

[0451] 3. In vitro filled AAV2 are able to infect and transduce HEK293 cells producing an observable signal when viewed with a microscope (Figure 11).

[0452] 4. Using the reporter assay, the present inventors can compare the potency of control AAV2 and in vitro filled AAV2 on two levels: a. On a capsid basis - in vitro filled AAVs are 2-3x more potent (Figure 13), this is due to a higher proportion of filled AAV particles b. On a per genome basis - in vitro filled AAVs are also 40-80% more potent (Figure 12). As viral genome quantity was matched between samples, and capsid quality is identical between samples, this effect can only be explained by differences in genome truncation. Genome truncation has been identified as a major concern by the FDA (Nguyen GN, 2021). The data implies more potent AAV demonstrate reduced DNA truncation, a significant advantage over current manufacturing technologies.

[0453] Example 4: Scalable, lysate based capsid filling - serotype expansion

[0454] Introduction

[0455] Example 1 demonstrated a method for using scalable and GMP-compatible techniques to enable in vitro AAV2 capsid filling for clinically relevant and drug development use.

[0456] Example 2 identified several conditions that significantly improve titers of in vitro capsid filling for AAV2. Although AAV2 is a popular serotype in pre-clinical and clinical settings, there are additional relevant serotypes, such as AAV5. AAV5 is considered the most divergent all the AAV serotypes (Issa et al., 2023). AAV5 also demonstrates the lowest seroprevalence of all the serotypes (Klamroth et al., 2022). Low preexisting immunity, and reduced risk of antibody cross reactivity with heterologous AAV serotypes, has made AAV5 a popular AAV serotype for therapeutic applications. However, the therapeutic potential of AAV5 has been limited by the absence of efficient and high quality methods for capsid filling.

[0457] The present inventors have found that presently disclosed in vitro capsid filling methods also allows for in vitro capsid filling of AAV5, hereby confirming that the presently disclosed in vitro capsid filling methods can be expanded to other AAV serotypes.

[0458] Equipment and materials

[0459] Equipment

[0460] Materials

[0461] Methods

[0462] Preparative procedures

[0463] Component generation

[0464] Plasmid generation pHelper plasmid and pAAV-GFP plasmid were sourced as described in Example 1 . In addition to this, a plasmid preparation encoding AAV5 capsid and Rep proteins was generated as described below. pAAV-RC5 (cat no.: VPK-425) was purchased from Cell Biolabs. This contained the AAV2 Rep gene and the AAV5 Cap gene. The annotated sequence is listed in ’’Sequence Overview” (SEQ ID NO: 28). pAAV-RC5 plasmid was transformed into NEB-DH5a cells by Synvolux BV (Leiden, The Netherlands), and amplified as described in Example 1 .

[0465] Cell lysate generation

[0466] VPC 2.0 cells were expanded in Viral Production Medium with GlutaMax. Incubation was at 8% CO2, 37°C, and 85% RH. The culture was diluted in pre-warmed medium to a concentration of 3 x 106VC / mL. For transfection the AAV-MAX Helper-Free AAV Production System guide was used (Gibco, 2023), with the following notes:

[0467] • Culture volume: 30mL (in 125mL unbaffled vented shake flask)

[0468] • VCD: 3 x 106VC / mL

[0469] • AAV Max enhancer: 300pL

[0470] • Viral plex complexation buffer volume: 3mL

[0471] • Transfection booster: 90pL

[0472] • Transfection reagent: 180pL

[0473] • pHelper (1.5 pg / pL): 18.4pL

[0474] • pAAV-RC5 (0.252 pg / pL): 68.9pL

[0475] • Complexation incubation time: 25 minutes

[0476] After 72 hours ,the culture was subjected to a 2-hour lysis with 3mL AAV Max lysis buffer supplemented with MgCh, at 37°C. Lysates were centrifuged at 13000xg, 15 minutes and supernatant was stored at - 20+-5°C.

[0477] Capsid AAV Packaging Matrix preparation

[0478] Inactive CAPM (lacking enzymes) medium was prepared by supplementing HEPES 30mM, pH 6.0 solution with the following (Table 18):

[0479] Table 18 Base buffer components

[0480] The following working solutions were used: • CPK solution (1000pg / mL): prepared as described in Example 1

[0481] • Clarified lysate as described in Example 1 .

[0482] • GFP plasmid (0.9pg / pL)

[0483] • ATP solution (200mM) In vitro AAV5 capsid filling

[0484] To activate CAPM medium, CPK solution was added to inactive CAPM medium.

[0485] The following conditions were prepared on ice (Tables 19-20):

[0486] Table 19 In vitro reaction mixture for filling AAV5 capsids j |

[0487] Table 20 In vitro reaction mixture control: no plasmid addition Reactions were incubated at 300 rpm, 39°C for 4 hours on a heat block and analyzed with qPCR as described in ’’Analytical procedures”.

[0488] Analytical procedures qPCR analysis was performed as described in Example 1 . Any results below the limit of quantification were considered 0. Results are shown in Figure 14. Figure 14 shows that the present in vitro capsid filling method achieve clinically significant titers (1 .1 e11 gc / mL) for AAV5.

[0489] Conclusion

[0490] The present inventors have successfully demonstrated that the presently disclosed in vitro capsid filling methods can be extended to other AAV serotypes, including the clinically relevant serotype (AAV5). The present in vitro capsid filling method achieves clinically significant titers (1 .1 e11 gc / mL). Example 5: Chemically defined capsid filling

[0491] Introduction

[0492] Example 1 demonstrated a method for using scalable and GMP-compatible techniques to enable in vitro AAV2 capsid filling for clinically relevant and drug development use.

[0493] Example 2 identified several conditions that significantly improve titers of in vitro capsid filling for AAV2.

[0494] Example 4 demonstrated that the method could be used for the most divergent AAV serotype (AAV5) thus illustrating the broad applicability of the method across all AAV serotypes.

[0495] Previous examples have used Rep proteins in a clarified cell lysate. In vitro filling using a purified stock of Rep proteins has several advantages, including:

[0496] • Significant impurity removal during protein purification, reducing purification burden post in vitro filling, minimizing drug substance loss.

[0497] • In vitro filling of AAV in crude lysate (Examples 1-4) may contain host cell-impurities. Impurity removal improves in vitro filling reaction control and optimization by using Rep concentrations as process parameters.

[0498] • Impurities present in Examples 1-4 may impact in vitro filling reaction kinetics. Using purified protein starting material will improve in vitro filling process control and optimization, by negating any possible interference of host cell components not required for in vitro filling.

[0499] • Increased process robustness and reproducibility due to chemically defined processing.

[0500] Equipment and materials

[0501] Equipment

[0502] Materials

[0503] Methods Preparative procedures

[0504] Component generation

[0505] Empty capsid generation

[0506] AAV5 plasmid and empty capsid generation was performed as described in Example 4. Purification techniques as adopted in Example 3.

[0507] Rep sequence design

[0508] Four Rep proteins are encoded in a single Rep gene in the wtAAV genome. The different proteins are expressed from a combination of alternate splicing and leaky start codons. In order to separately express Rep40, Rep52, Rep 68, Rep78 as four separate proteins, the amino acid sequences had to be extrapolated from Rep. Once determined, each Rep protein sequence was combined with an N-terminal cleavable SUMO fusion domain containing a purification tag, improving solubility, and a C-terminal ‘c-tag’ (aa sequence: EPEA) for purification (sequences in ’’Sequence Overview” SEQ ID NOs: 29-32). Thus four plasmid designs were created, one for each Rep protein. For each Rep protein a new cell bank was created. Methods described in sections “Transformation and cell bank”, “Production”, “Cell lysis and purification” and “Dialysis and proteolytic activation” were run four times in parallel, one for each of the four rep proteins.

[0509] Transformation and cell bank

[0510] The codon-optimized synthetic gene sequence of the target gene coding for Hise-SUMO-Rep-EPEA was transformed into 25 pL competent BL21 (DE3) strain by adding 0.5 pL 50 ng / pL of target plasmid DNA. The mixture is flicked to thoroughly mix and incubated on ice for 30 min. Cells are then heat shocked for exactly 10 seconds at 42°C and placed on ice for 5 min. 475 pL room temperature SOC medium was added, and the cells was placed at 37°C for 60 min with 250 rpm shaking in a benchtop shaker. 200 pL was then spread onto a selective plate consisting of LB medium containing 50 mg / L kanamycin as selection marker and incubated overnight at 37°C. A scrape of transformed E. co / / -cultures from selective plates was used to inoculate 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. The culture was incubated overnight in an incubator with shaking at 250 rpm at 37°C. The following morning, 25 mL autoclaved LB medium supplemented with 50 mg / L kanamycin was inoculated with 250 pL of the overnight culture and placed at 37°C with 250 rpm shaking in an incubator. When ODeoo reached 1-2, sterilized glycerol was added to a final concentration of 15% and shaken until homogenous. The medium was aliquoted in 10x1 mL labelled vials and stored at -70°C as MCB. The presence of the full-length Rep fusion protein was confirmed by SDS-PAGE.

[0511] Production

[0512] To scale up expression, a scrape from 1 MCB vial of each of the four constructs was used to inoculate a 15 mL falcon tube with 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. Cells were cultured overnight in a shaking incubator at 37.0°C with 250 rpm shaking. The following day, 4 x 2L sterile baffled shake flasks with 500 mL 2xYT medium supplemented with 50 mg / L kanamycin was inoculated with 5 mL of the overnight culture. Flasks were placed in a shaking incubator at 250 rpm at 37.0°C for approximately 3 hours. When the ODeoo reached -0.8-1 .2, cells were induced by adding IPTG to a final concentration of 1 mM. The shake flasks were incubated overnight at 18°C in the same incubator and same shaking. The following morning, cells were pelleted by centrifugation at 15,000g for 15 minutes at 4°C using a fixed-angel rotor. Supernatants were discarded and pellets were stored at -20°C until further use. Cell lysis and purification

[0513] Cells were resuspended in 15 mL lysis buffer (20 mM Tris HCI pH 8.0, complete protease inhibitor, 5 mM DTT, 7 U / ml Denarase) in a 50 mL falcon tube and lysed by sonication for 10 minutes with pulses of 2 seconds on and 4 seconds off at an amplitude of 70%, keeping the sample in an ice bath to prevent heating. Insoluble material was removed by centrifugation at 15,000g for 15 minutes using a fixed angle rotor at 4°C. The supernatant was sterile filtered through a 0.2 pm filter and loaded onto a CaptureSelect C-tagXL MiniChrom column equilibrated in equilibration buffer (100 mM Tris HCI pH 8.0). The column was washed with a wash buffer (50 mM Tris HCI pH 8.0, 150 mM NaCI) and the protein of interest was eluted using an elution buffer (50 mM Tris HCI pH 8.0, 2 M MgCh). SDS-PAGE (Figure 15 and Figure 16) and Western blotting (data not shown) using an anti-AAV2 Replicase mouse monoclonal antibody was performed to positively identify the presence, purity, and identity of the proteins of interest.

[0514] Dialysis and proteolytic activation

[0515] Purified samples were dialyzed against PBS pH 7.4 containing 10% glycerol using a 10 kDa cutoff. Dialysis was performed at 5°C while slowly stirring. After at least 3 hours, the dialysis buffer was exchanged with fresh dialysis buffer and continued overnight. The following morning, the samples were centrifuged at 15,000g for 10 min at 5°C in a benchtop centrifuge. SUMO protease cleavage was carried out under these conditions: all target protein was adjusted to 10 pM in a 500 pL setup and incubated for 16 hours at 5°C and at room temperature with 0.1 U / pL SUMO protease and 1 mM DTT. Cleavage was verified by SDS-PAGE (Figure 17 and Figure 18) and Western blot (data not shown) using an anti-AAV2 Replicase mouse monoclonal antibody.

[0516] Capsid AAV Packaging Matrix preparation

[0517] Inactive CAPM (lacking enzymes) medium was prepared by supplementing HEPES 30mM, pH 6.0 solution with the following (Table 21):

[0518] Table 21 Base buffer components

[0519] The following working solutions were used:

[0520] • CPK solution (1000pg / mL): prepared as described in Example 1

[0521] • Clarified lysate as described in Example 1 . • GFP plasmid (0.9pg / pL)

[0522] • ATP solution (200mM)

[0523] In vitro AAV5 capsid filling

[0524] The following conditions were prepared on ice (Tables 22-26):

[0525] Table 22. AAV5 in vitro filled particles | I I | | | | I | I j

[0526] *10pM of SUMO protease treated Rep protein.

[0527] Table 23. Control without Rep I

[0528] Table 24. Control without pDNA

[0529] Table 25. Control buffer only

[0530] Reactions were incubated at 300 rpm, 39°C for 4 hours on a heat block and analyzed with qPCR as described in “Analytical procedures - qPCR”. Results shown in Figure 19.

[0531] Analytical procedures qPCR qPCR analysis was performed as described in Example 1 . Any results below the limit of quantification were considered 0. Data shown in Figure 19.

[0532] SDS page

[0533] Expression, purification and SUMO enzymatic cleavage of Rep was assessed using SDS-Page. Data shown in Figure 15, Figure 16, Figure 17 and Figure 18.

[0534] Conclusion

[0535] The present Example describes: 1 . The novel method of in vitro filling of AAV5 using purified Rep proteins, the first demonstration of chemically defined capsid filling (Figure 18)

[0536] 2. The successful development of novel fusion proteins that contain: a. An AAV Rep domain (’’Sequence Overview” SEQ ID NOs: 29-32) b. A cleavable SUMO domain that enhances expression and purification (’’Sequence Overview” SEQ ID NOs: 29-32)

[0537] 3. The expression of individual AAV rep proteins in the prokaryotic microbial system, Escherichia coli (Figure 15 and Figure 16).

[0538] 4. Methods for SUMO protease digestion liberating Rep proteins of SUMO fusion element, where time and temperature are critical parameters (Figure 17 and Figure 18).

[0539] Due to supporting data in Examples 1-4, the method described here for AAV5 can be applied to all AAV serotypes.

[0540] Example 6: Capsid assembly and stoichiometry control through precipitation Introduction

[0541] Adeno-associated virus (AAV) consists of a single-stranded DNA genome of 4.7 kilobases packed in a non-enveloped capsid of 60 proteins. AAV serotype 2 (AAV2) is the best-studied member of the genus which currently comprises several serotypes possessing diverging tropism (Issa et al., 2023). The AAV genome consists of three open reading frames (ORFs) that encode the four Rep proteins required for genome assembly, the assembly-activating protein that promotes assembly of the capsid, and the three capsid proteins VP1 , VP2 and VP3. The major capsid protein VP3 assembles the icosahedral capsid, while the minor capsid protein VP1 and VP2 are required for intracellular trafficking, escape, nuclear localization and genome release. All VPs share the VP3 domain and are assembled with the relative abundance of VP1 / 2 / 3 as 1 :1 :10 (Worner et al., 2021). Recombinant AAV (rAAV) can be produced in HEK293 cells using a triple-plasmid method or in Sf9 cells using baculovirus. These production methods suffer from considerable problems with scalability and product heterogeneity (Samulski et al., 2014).

[0542] Le et al., 2019 has shown recombinant production of AAV2 VP3 protein as inclusion bodies in Escherichia coli and subsequent refolding (Le et al., 2019). While this proof-of-principle study makes some advances, the primary application remains rather limited, i.e., it only involves the production viruslike particles (VLPs) used in vaccination and therefore only makes use of the major capsid protein. The present inventors have discovered novel in vitro capsid assembly methods. These improved methods makes it possible to incorporate VP1 and VP2 in a titratable manner and expand applicability by including different serotypes. The present inventors produce all three capsid proteins from AAV5 as inclusion bodies in E. coli and, after solubilization, purify them to a degree compatible with refolding. The ability to refold AAV5 VP3 is first screened in a host of different buffers. The present inventors show that AAV5 VP3-only capsid can indeed be formed under certain conditions. Incubating for shorter periods improves titer. The present inventors then proceed to purify AAV5 VP1 , AAV5 VP2 and AAV5 VP3 further. Mixing these preparations at different ratios results in the formation of capsid particles, which can be purified using the assembled capsid-specific AAVX purification resin. The results described here offer control of the stoichiometry of the different coat proteins within the viral capsid. Equipment and materials

[0543] Equipment

[0544] Materials

[0545] Methods

[0546] Preparative procedures

[0547] Transformation and protein production

[0548] The following expression constructs were transformed into E. coli BL21 (DE3) and plated out on kanamycin selective growth plates (LB agar):

[0549] • pET29-AAV5-VP1

[0550] • pET29-AAV5-VP2

[0551] • pET29-AAV5-VP3

[0552] The protein sequences of the proteins of interest produced from these constructs can be found in ’’Sequence Overview” (SEQ ID NOs: 2, 20, 33). One colony was taken from these plates after growth overnight at 37°C and was used to inoculate 5 ml LB media containing 50 pg mL-1kanamycin. These were incubated overnight (16 hours) at 37°C shaking at 250 rpm. The next morning, six sterile 2L baffled flasks were prepared with 500 mL 2YT media and inoculated with 5 ml overnight culture. Cultures were grown at 37°C shaking at 250 rpm to an OD of 1 .0 whereupon 0.5 mL 1 M IPTG was added. Cultures were grown overnight at 37°C shaking at 250 rpm. Cells were harvested the next day at 10000g for 30 minutes in 500 mL sterile centrifuge tubes and pellets were stored at -20°C until further use.

[0553] Inclusion body preparation and solubilization

[0554] Cells were resuspended in a 50 mL tube on ice in 40 mL lysis buffer (100 mM Tris Ci, pH 8.0, 5 mM EDTA pH 8.0, complete protease inhibitor, 5 mM DTT) after which the volume was adjusted to 50 mL. Cells were lysed by sonication for 10 min in a 100 mL glass beaker placed in an ice bath, with pulses of 2 sec on and 4 sec off, at an amplitude of 70%, keeping the sample in an ice bath to prevent heating. The lysate was centrifuged at 8000g for 15 minutes at 10°C and the supernatant was discarded. The pellet was washed three times with wash buffer 1 (100 mM Tris Ci pH 8.0, 5 mM EDTA pH 8.0, 2 M urea, 2% (w / v) Tergitol™ 15-S-9, 5 mM DTT) and twice with wash buffer 2 (100 mM Tris Ci pH 8.0, 5 mM EDTA pH 8.0, 2 M urea, 5 mM DTT). Inclusion bodies were solubilized while rotating overnight in solubilization buffer (20 mM Tris Ci pH 8.0, 8 M urea, 5 mM DTT). The next morning, any insoluble residue was removed by centrifugation at 15000g for 30 minutes at 4°C. The supernatant was kept at 4°C until further processing.

[0555] Protein purification

[0556] AAV5 VP1 , AAV5 VP2, and AAV5 VP3 were purified using ion exchange and size exclusion chromatography (SEC). In short, solubilized inclusion bodies were loaded onto a cation exchange column equilibrated in 20 mM Tris Ci pH 8.0, 8 M urea and the protein of interest was eluted with a gradient of 20 mM Tris Ci pH 8.0, 8 M urea, 1 M NaCI over 20 column volumes. The AAV5 VP3 protein was then used for refolding experiments. The final concentration of AAV5 VP3 was 0.56 g I-1. To increase purity, samples were concentrated to 2 mL using a 10 kDa spin concentrator and 0.5 mL was loaded onto a Superdex 200 SEC column equilibrated in 20 mM Tris Ci pH 8.0, 8 M urea, 150 mM NaCI. The mobile phase consisted of 20 mM Tris Ci pH 8.0, 8 M urea, 150 mM NaCI. The peaks containing the proteins of interest were collected and used in subsequent experiments.

[0557] Capsid purification

[0558] AAVX resin was equilibrated 3 times with 1 volume of 50 mM Tris Ci pH 8.0, 150 mM NaCI and resuspended to a 62.5% slurry. Samples containing refolded capsid particles were incubated with 400 pL equilibrated slurry resin for 1 hour at 4°C while mixing. After pelleting the resin and discarding the supernatant, the resin was resuspended in 500 pL wash buffer 1 (50 mM Tris Ci pH 9.0, 1 M NaCI) and incubated for 5 minutes at 4°C. This process was repeated with wash buffer 2 (50 mM Tris Ci pH 9.0) and mQ water. Finally, elution of capsid particles was performed by incubating the pelleted resin with 200 pL elution buffer (100 mM glycine pH 2.5, 50 mM NaCI) for 5 minutes at 4°C. The resin was pelleted at 1000g for 5 minutes at 4°C in between equilibration, wash, and elution steps.

[0559] Refolding of AAV5 VP3 into capsid particles

[0560] Refolding properties of AAV5 VP3 were screened by rapid dilution (100x) into 17 different buffers (listed below) and incubation for 16 hours at 4°C and 22°C. All buffers were shown to support the formation of capsid particles, i.e. , allowed for capsid assembly. Buffer 11 was shown to support formation of capsid particles particularly well. Subsequent refolding experiments in this buffer were performed for 1 hour and 16 hours at room temperature (22°C). Buffers:

[0561] Buffer 1 : 50 mM MES pH 6.0, 9.6 mM NaCI, 0.4 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.75 M Guanidine HCI, 0.5% Triton X-100, 0.1 mM DTT

[0562] Buffer 2: 50 mM MES pH 6.0, 9.6 mM NaCI, 0.4 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.5 M arginine, 0.05% polyethylene glycol 3,550, 0.1 mM GSH, 0.01 mM GSSH

[0563] Buffer 3: 50 mM MES pH 6.0, 9.6 mM NaCI, 1 mM EDTA, 0.4 mM KCI, 0.4 M sucrose, 0.75 M Guanidine HCI, 0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM DTT

[0564] Buffer 4: 50 mM MES pH 6.0, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.5 M arginine, 0.5% Triton X-100, 0.1 mM GSH, 0.01 mM GSSH

[0565] Buffer 5: 50 mM MES pH 6.0, 240 mM NaCI, 10 mM KCI, 1 mM EDTA, 0.4 M sucrose, 0.75 M Guanidine HCI, 0.1 mM DTT

[0566] Buffer 6: 50 mM MES pH 6.0, 240 mM NaCI, 10 mM KCI, 1 mM EDTA, 0.5 M arginine, 0.4 M sucrose, 0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH, 0.01 mM GSSH

[0567] Buffer 7: 50 mM MES pH 6.0, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.75 M Guanidine HCI, 0.05% polyethylene glycol 3,550, 0.1 mM DTT

[0568] Buffer 8: 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.4 M sucrose,

[0569] 0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH, 0.01 mM GSSH

[0570] Buffer 9: 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 1 mM EDTA, 0.5 M arginine, 0.75 M

[0571] Guanidine HCI, 0.05% polyethylene glycol 3,550, 0.1 mM DTT

[0572] Buffer 10: 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.5 M arginine, 0.4 M sucrose, 0.75 M Guanidine HCI, 0.1 mM GSH, 0.01 mM GSSH

[0573] Buffer 11 : 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 1 mM EDTA, 0.5% Triton X-100, 0.1 mM DTT

[0574] Buffer 12: 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 1 mM EDTA, 0.05% polyethylene glycol 3,550, 0.1 mM GSH, 0.01 mM GSSH

[0575] Buffer 13: 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 1 mM EDTA, 0.5 M arginine, 0.75 M

[0576] Guanidine HCI, 0.5% Triton X-100, 0.1 mM DTT

[0577] Buffer 14: 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.5 M arginine, 0.4 M sucrose, 0.75 M Guanidine HCI, 0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH, 0.01 mM GSSH

[0578] Buffer 15: 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.4 M sucrose, 0.1 mM DTT

[0579] Buffer 16: 20 mM Tris-CI, 8 M urea, pH 8.0

[0580] Buffer 17: 500 mM NaCI, 100 mM KCI, 10 mM Na2HPO4, 10 mM KH2PO4, pH 8.0 Stoichiometric control

[0581] Partially purified AAV5 VP1 , AAV5 VP2 and AAV5 VP3 proteins were mixed in ratios of 1 :1 :20, 1 :1 :1 , and 10:10:1 based on densitometric image analysis of an SDS PAGE purity gel. The volume was adjusted to 5 mL 20 mM Tris Ci, 8 M urea, pH 8.0, 1 mM DTT and the sample was dialyzed against 500 mM NaCI, 100 mM KCI, 10 mM Na2HPO4, 10 mM KH2PO4, pH 8.0 using a 10 kDa cutoff membrane. Dialysis was performed for 3 hours at 4°C while stirring, whereupon the buffer was replaced with fresh dialysis buffer. Dialysis was continued overnight (16 hours) at 4°C while stirring. The next morning, samples were centrifuged at 15000g for 20 minutes at 4°C. Capsid particles were purified as described above before further analysis.

[0582] Analytical procedures

[0583] SDS page and Western blotting

[0584] Identity of AAV5 VP1 , AAV5 VP2, and AAV5 VP3 proteins and stoichiometric control of capsid particles was confirmed by SDS page and western blotting using an anti-AAV antibody (Figure 20).

[0585] Capsid ELISA

[0586] AAV5 capsid formation was confirmed using the AAV5 Xpress ELISA kit .

[0587] Conclusion

[0588] The present Examples shows:

[0589] • AAV5 VP1 , AAV5 VP2, and AAV5 VP3 can be produced as inclusion bodies in E. coll (Figure 20).

[0590] • AAV5 VP1 , AAV5 VP2, and AAV5 VP3 can be purified to a level compatible with refolding (Figure 21 , Figure 24.)

[0591] • Buffer screening revealed specific conditions that enable VPs to refold into AAV5 capsids (Figure 22). All of the tested buffers enabled VPs to refold into AAV5 capsids.

[0592] • The titer of AAV5 VP3 only capsids obtained in AAV5 particle ELISA is 1 .7-5.3»108capsids mL-1and is temperature dependent and time dependent (Figure 23).

[0593] • Simultaneous, mixed refolding of VP1 ; VP2; and VP3, followed by affinity-based capsid purification revealed capsid populations with different VP1 :VP2:VP3 stoichiometries (Figure 25). This demonstrates that the method can be used to control capsid VP stoichiometry.

[0594] Example?: Capsid assembly through SUMO proteolysis Introduction

[0595] The present inventors have found a novel way of assembling AAV capsids. One possible route to achieve this is using cleavable fusion proteins that enable capsid protein stoichiometry control further downstream in a cell-free environment. The fusion proteins contain several elements of interest: a His-tag for purification purposes, a SUMO fusion protein, VP proteins, a thrombin cleavage site, SUMO fusion protein, and a C-tag (EPEA) for purification purposes (SEQ ID NO: 34). SUMO proteins enable (1) increased solubility of protein of interest (VP) and (2) steric hindrance between capsid proteins to prevent or minimize spontaneous intracellular assembly. Each SUMO protein has a cleavable amino acid sequence towards its C-terminus. Two SUMO moieties are fused N- and C-terminally to VP3 protein of AAV2.

[0596] This example describes (1) P. pastoris-based expression of the gene coding for His-SUMO-AAV2-VP3- SUMO-EPEA and associated purification methods; and (2) induced assembly of VP3 proteins into capsids upon proteolytic cleavage.

[0597] Equipment and materials

[0598] Equipment

[0599] Materials

[0600] Methods

[0601] Preparative procedures

[0602] Construct selection

[0603] The codon-optimized synthetic gene sequence of the target gene coding for His-SUMO-AAV2-VP3- SUMO-EPEA was cloned via EcoRI / Notl restriction into an appropriately digested expression plasmid pPZ carrying a wild type PAOX1 promoter. After transformation into E. coli TOP1 OF' cells, recultivation of transformants and plasmid preparation, correct insertion of the target gene was checked by restriction pattern analysis, and authenticity of the gene sequence was confirmed by sequencing (LGC Genomics, Berlin, Germany). The plasmid was linearized using Sacl endonuclease and desalted for transformation into P. pastoris. DNA concentration was determined by spectrophotometric measurement and adjusted to be roughly 1 pg pL-1.

[0604] Electroporation was carried out using competent cells of basic production strain CBS7435 MutS (genotype A aox1 ; phenotype MutS) applying a standard procedure and standard equipment for electroporation. After regeneration at 28°C, preparations were plated on agar plates containing the antibiotics Zeocin and Geneticin. Single colonies (8 per transformation) were picked from transformation plates into single wells of a 96-deep well plate filled with optimized cultivation media. Remaining wells were inoculated with a mock strain. After an initial growth phase to generate biomass, expression was induced by addition of an optimized liquid mixture containing a defined concentration of methanol. At defined points of time, further induction with methanol was performed. After a total of 50 hours from the initial methanol induction, cells were collected by centrifugation. The presence of the full-length AAV2 VP3 fusion protein was confirmed by Western blotting. A master cell bank (MCB) in glycerol was prepared of the clones expressing the fusion protein. Production

[0605] To scale up expression, four vials of the MCB were defrosted and added in 4 x 2L flasks containing 250 mL BMGY medium supplemented with biotin. Flasks were placed in the shaker at 250 rpm at 30°C for approximately 18 hours. When the OD reached ~30, cells were pelleted by centrifugation at 3200g for 20 minutes at 4°C. After the supernatant with spent media was discarded, cells were resuspended in 250 mL BMGY medium supplemented with biotin. When the OD reached -100, 1 .5% (v / v) MeOH was added. After 48 hours of incubation shaking at 250 rpm at 30°C, cells were harvested by centrifugation at 15000g for 20 minutes at 4°C. Pellets were stored at -20°C until further use.

[0606] Cell lysis and purification

[0607] Cells were resuspended in 50 mL lysis buffer (20 mM Tris Ci pH 8.0, 2 mM MgCh, complete protease inhibitor, 5 mM DTT, 7 U / ml Denerase) and lysed by sonication for 3 minutes with pulses of 20 seconds on and 40 seconds off at an amplitude of 90%, keeping the sample in an ice bath to prevent heating. Directly after, 0.1 % (v / v) Tergitol 15-S-9 was added, and the sample was incubated on ice for 20 minutes. Insoluble material was removed by centrifugation at 15000g for 15 minutes at 4°C. The supernatant was loaded onto a CaptureSelect C-tagXL MiniChrom column equilibrated in binding buffer (100 mM Tris Ci pH 8.0). The column was washed with wash buffer 1 (100 mM Tris Ci pH 8.0, 150 mM NaCI) and the protein of interest was eluted using elution buffer (50 mM Tris Ci pH 8.0, 2 M MgCh). SDS PAGE and Western blotting using an anti-AAV antibody was performed to positively identify the presence and identity of the proteins of interest.

[0608] Induced assembly by proteolytic activation

[0609] After dialysis overnight into PBS using a 10 kDa cutoff membrane, SUMO- and thrombin cleavage was carried out simultaneously under these conditions: all target protein was incubated for 16 hours overnight at room temperature (22°C) with 12 pL 0.5 U / pL Thrombin and 2 pL 10 U / pL SUMO protease 2.

[0610] Analytical procedures

[0611] Western blotting

[0612] Cleavage of the proteins of interest was confirmed by SDS PAGE and western blotting (Figure 26) using an anti-AAV antibody.

[0613] Capsid ELISA

[0614] After concentrating a hundredfold using a 100 kDa spin concentrator, formation of capsid particles was assayed with an AAV2-specific capsid ELISA at 10x and 100x dilutions. Capsid titers were calculated using a best fit four parameter logistic equation (Figure 27). Conclusion

[0615] The present Example describes:

[0616] 1 . The successful development of a novel fusion protein that contains: a. An AAV VP domain b. Two different, cleavable, sterically inhibiting domains preventing spontaneous selfassembly and enabling increased capsid assembly process control c. Two different purification tags, enabling the use of pure full-length fusion proteins for in vitro assembly reactions

[0617] 2. The expression of AAV capsid proteins in the eukaryotic microbial system, Pichia pastoris. This unlocks a wide array of expression features and process controls limited strictly to methylotrophic yeasts, such as control of protein glycosylation and methanol-based induction.

[0618] 3. Methods for SUMO digestion and resultant capsid assembly, where time and temperature are critical parameters.

[0619] 4. Method development for sterically controlled in vitro AAV capsid assembly for high capsid titers.

[0620] The methods described here can also be applied to enable capsid stoichiometric VP control as described in Example 6.

[0621] Example 8: Chemically Defined High Fill Rate (AAV5)

[0622] Example 5 demonstrated a novel method of in vitro filling of AAV5 using purified Rep proteins, the first demonstration of chemically defined capsid filling. Methods were provided for the successful development of novel fusion proteins that contain an AAV Rep domain and a cleavable SUMO domain that enhances expression and purification. These fusion proteins can be individually expressed in the prokaryotic microbial expression system, Escherichia coli, and the Rep moiety can be liberated by SUMO protease digestion, where time and temperature might be critical parameters.

[0623] This research laid an essential groundwork for transitioning in vitro filling technology to an industrially feasible process. The present inventors further improved the in vitro filling technology. Firstly, it was determined how different combinations of Rep proteins can influence capsid filling rates. Secondly, previously used Rep moieties were prepared with cleavable SUMO-histidine tags to simplify purification. This technique necessitates the use of imidazole and cleavage enzymes, rendering it less translatable for clinical applications and Good Manufacturing Practice (GMP) production. Therefore, a GMP-compatible Rep construct had to be developed. The following example demonstrates achieving an industrially viable in vitro filling process by (1) maximizing AAV fill rates with (2) GMP-compatible methods. This was accomplished by:

[0624] • Examining optimal combinations of Rep proteins

[0625] • Expanding the range of expression-enhancing purification tags • Further engineering a Rep protein to eliminate the need for his-tags (GMP compatibility) and cleavable elements

[0626] • Achieving 100% observed fill rates of AAV

[0627] Equipment and materials Equipment

[0628] Materials

[0629] Methods

[0630] Preparative procedures

[0631] Component generation Empty capsid generation

[0632] AAV5 plasmid and empty capsid generation was performed as described in Example 4. Purification techniques as adopted in Example 3.

[0633] SUMO-Rep production

[0634] SUMO-Rep sequence design In the wtAAV genome, the rep gene, through the use of two promoters and alternative splicing, encodes four regulatory proteins that are dubbed Rep78, Rep68, Rep52 and Rep40. In order to separately express Rep40, Rep52, Rep 68, Rep78 as four separate proteins, the amino acid sequences had to be extrapolated from Rep. Once determined, each Rep protein sequence was combined with an N-terminal cleavable SUMO fusion domain containing a purification tag, improving solubility, and a C-terminal ‘c-tag’ (aa sequence: EPEA) for purification (sequences of SEQ ID NOs: 36-39 listed in ’’Sequence Overview”). Thus, four plasmid designs were created, one for each Rep protein. For each Rep protein a new cell bank was created. Methods described in sections below were run four times in parallel, one for each of the four rep proteins.

[0635] Transformation and cell bank

[0636] The codon-optimized synthetic gene sequence of the target gene coding for Hise-SUMO-Rep-EPEA was transformed into 25 pL competent BL21 (DE3) strain by adding 0.5 pL 50 ng / pL of target plasmid DNA. The mixture was flicked to thoroughly mix and incubated on ice for 30 min. Cells were then heat shocked for exactly 10 seconds at 42°C and placed on ice for 5 min. 475 pL room temperature SOC medium was added, and the cells was placed at 37°C for 60 min with 250 rpm shaking in a benchtop shaker. 200 pL was then spread onto a selective plate consisting of LB medium containing 50 mg / L kanamycin as selection marker and incubated overnight at 37°C. A scrape of transformed E. coli-cultures from selective plates was used to inoculate 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. The culture was incubated overnight in an incubator with shaking at 250 rpm at 37°C. The following morning, 25 mL autoclaved LB medium supplemented with 50 mg / L kanamycin was inoculated with 250 pL of the overnight culture and placed at 37°C with 250 rpm shaking in an incubator.

[0637] When ODeoo reached 1-2, sterilized glycerol was added to a final concentration of 15% and shaken until homogenous. The medium was aliquoted in 10x1 mL labelled vials and stored at -70°C as MCB. The presence of the full-length Rep fusion protein was confirmed by SDS-PAGE.

[0638] Production

[0639] To scale up expression, a scrape from 1 MCB vial of each of the four constructs was used to inoculate a 15 mL falcon tube with 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. Cells were cultured overnight in a shaking incubator at 37.0°C with 250 rpm shaking. The following day, 4 x 2L sterile baffled shake flasks with 500 mL 2xYT medium supplemented with 50 mg / L kanamycin was inoculated with 5 mL of the overnight culture. Flasks were placed in a shaking incubator at 250 rpm at 37.0°C for approximately 3 hours. When the ODeoo reached -0.8-1 .2, cells were induced by adding IPTG to a final concentration of 1 mM. The shake flasks were incubated overnight at 18°C in the same incubator and same shaking. The following morning, cells were pelleted by centrifugation at 15,000g for 15 minutes at 4°C using a fixed-angel rotor. Supernatants were discarded and pellets were stored at -20°C until further use. Cell lysis and purification

[0640] Cells were resuspended in 15 mL lysis buffer (20 mM Tris HCI pH 8.0, complete protease inhibitor, 5 mM DTT, 7 U / ml Denarase) in a 50 mL falcon tube and lysed by sonication for 10 minutes with pulses of 2 seconds on and 4 seconds off at an amplitude of 70%, keeping the sample in an ice bath to prevent heating. Insoluble material was removed by centrifugation at 15,000g for 15 minutes using a fixed angle rotor at 4°C. The supernatant was sterile filtered through a 0.2 pm filter and loaded onto a CaptureSelect C-tagXL MiniChrom column equilibrated in equilibration buffer (100 mM Tris HCI pH 8.0). The column was washed with a wash buffer (50 mM Tris HCI pH 8.0, 150 mM NaCI) and the protein of interest was eluted using an elution buffer (50 mM Tris HCI pH 8.0, 2 M MgCh). SDS-PAGE and Western blotting using an anti-AAV2 Replicase mouse monoclonal antibody was performed to positively identify the presence, purity, and identity of the proteins of interest (data not shown).

[0641] Dialysis and proteolytic activation

[0642] Purified samples were dialyzed against PBS pH 7.4 containing 10% glycerol using a 10 kDa cutoff. Dialysis was performed at 5°C while slowly stirring. After at least 3 hours, the dialysis buffer was exchanged with fresh dialysis buffer and continued overnight. The following morning, the samples were centrifuged at 15,000g for 10 min at 5°C in a benchtop centrifuge. SUMO protease cleavage was carried out under these conditions: all target protein was adjusted to 10 pM in a 500 pL setup and incubated for 16 hours at 5°C and at room temperature with 0.1 U / pL SUMO protease and 1 mM DTT. Cleavage was verified by SDS-PAGE and Western blot (data not shown) using an anti-AAV2 Replicase mouse monoclonal antibody. Cleavage was verified by SDS-PAGE (data not shown) and Western blot (data not shown) using an anti-AAV2 Replicase mouse monoclonal antibody.

[0643] MBP-Rep78 production

[0644] MBP-Rep78 sequence design

[0645] In the wtAAV genome, the rep gene, through the use of two promoters and alternative splicing, encodes four regulatory proteins that are dubbed Rep78, Rep68, Rep52 and Rep40. In order to separately express Rep78 as a separate protein, the amino acid sequences had to be extrapolated from Rep. Once determined, the Rep protein sequence was combined with an N-terminal cleavable MBP fusion domain, which improves solubility and acts as an affinity purification tag (sequence of SEQ ID NO: 35 listed in ’’Sequence Overview”). An expression plasmid was created, harboring the coding sequence of this fusion protein. The plasmid was transformed into an Escherichia coli expression strain and a new cell bank was created. Methods described below were used to purify the fusion protein to homogeneity. Transformation and cell bank

[0646] The codon-optimized synthetic gene sequence of the target gene coding for MBP-Rep78 was transformed into 25 pL competent BL21 (DE3) strain by adding 0.5 pL 50 ng / pL of target plasmid DNA. The mixture is flicked to thoroughly mix and incubated on ice for 30 min. Cells are then heat shocked for exactly 10 seconds at 42°C and placed on ice for 5 min. 475 pL room temperature SOC medium was added, and the cells was placed at 37°C for 60 min with 250 rpm shaking in a benchtop shaker. 200 pL was then spread onto a selective plate consisting of LB medium containing 50 mg / L kanamycin as selection marker and incubated overnight at 37°C. A scrape of transformed E. coli-cultures from selective plates was used to inoculate 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. The culture was incubated overnight in an incubator with shaking at 250 rpm at 37°C. The following morning, 25 mL autoclaved LB medium supplemented with 50 mg / L kanamycin was inoculated with 250 pL of the overnight culture and placed at 37°C with 250 rpm shaking in an incubator. When ODeoo reached 1-2, sterilized glycerol was added to a final concentration of 15% and shaken until homogenous. The medium was aliquoted in 10x1 mL labelled vials and stored at -70°C as MCB.

[0647] Production

[0648] To scale up expression, a scrape from 1 MCB vial was used to inoculate a 15 mL falcon tube with 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. Cells were cultured overnight in a shaking incubator at 37°C with 250 rpm shaking. The following day, a 2L sterile baffled shake flasks with 500 mL autoinduction medium with 50 mg / L kanamycin was inoculated with 5 mL of the overnight culture. Flasks were placed in a shaking incubator at 250 rpm at 37.0°C for approximately 3 hours. When the ODeoo reached ~1 .0-1 .2, the temperature was lowered to 18°C and the shake flasks were incubated overnight in the same incubator and at the same shaking speed. The following morning, cells were pelleted by centrifugation at 15,000g for 15 minutes at 4°C using a fixed-angle rotor. Supernatants were discarded and pellets were stored at -20°C until further use.

[0649] Cell lysis and purification

[0650] Cells were resuspended in 200 mL lysis buffer (100 mM Tris HCI pH 8.0, complete protease inhibitor, 5 mM DTT, 2 mM MgCh, 10.5 U / ml Denarase, 1 mM EDTA) and lysed by sonication for 10 minutes with pulses of 2 seconds on and 4 seconds off at an amplitude of 70%, keeping the sample in an ice bath to prevent heating. Insoluble material was removed by centrifugation at 15,000g for 15 minutes using a fixed angle rotor at 4°C. The supernatant was sterile filtered through a 0.2 pm filter and loaded onto a MBPTrap HP column equilibrated in equilibration buffer (20 mM Tris HCI pH 7.4, 200 mM NaCI, 1 mM EDTA, 1 mM DTT). The column was washed with 5 column volumes of equilibration buffer and the protein of interest was eluted using elution buffer (20 mM Tris HCI pH 7.4, 200 mM NaCI, 1 mM EDTA, 1 mM DTT, 10 mM maltose). The eluate containing the protein of interest was loaded onto a HiTrap Q HP column equilibrated in equilibration buffer. The column was washed with 2 column volumes of equilibration buffer and elution was performed by a linear gradient of 10 column volumes to 25% elution buffer and a step elution with 5 column volumes of elution buffer (20 mM Tris HCL pH 7.4, 2M NaCI, 1 mM DTT). The flow through fractions containing the protein of interest were pooled and concentrated to 10 mL using spin concentrators (4000g, 4°C). The protein of interest was finally polished size exclusion chromatography using a Superose 6 column. Two runs were performed with a 5 mL load on a column equilibrated in PBS, 10% glycerol (w / v). The protein of interest eluted at a retention time of ~84 mL and corresponding fractions were pooled and concentrated to 5 mL by spin concentrators. The presence, purity, and identity of MBP-Rep78 was determined by SDS-PAGE (Figure 29) and Western blotting using an anti-AAV2 Replicase mouse monoclonal antibody (Figure 30). The protein was flash frozen in N2 (I) and stored at - 70°C.

[0651] In vitro filling buffer preparation 100 mM creatine phosphate was prepared in 30 mM HEPES, pH 6.0 and supplemented with the following components.

[0652] Table 27 Base buffer components

[0653] The following working solutions and components were used:

[0654] • CPK solution (1000 pg / mL); prepared as described in Example 1

[0655] • GFP plasmid (0.9 pg / pL); prepared as described in Example 1

[0656] • ATP solution (100 mM)

[0657] In vitro AA V5 capsid filling

[0658] To generate the data presented in Figure 28, the following components were thawed on ice.

[0659] Table 28. Component list AAV5 in vitro filled particles I j | | 1 | | I |

[0660] To generate the data presented in Figure 28, the following reactions were prepared.

[0661] Table 29. AAV5 particle in vitro filling reaction volume (pl)

[0662] Figure 28A shows AAV titers determined by qPCR of chemically defined in vitro filling reactions, using different combinations of recombinant Rep proteins.

[0663] Figure 28B shows AAV fill rates determined by capsid ELISA and qPCR of chemically defined in vitro filling reactions, using different combinations of recombinant Rep proteins.

[0664] To generate the data presented in Figure 31 , the following reaction was prepared on ice.

[0665] Table 30. AAV5 in vitro filled particles I j ) | | | I i |

[0666] Reactions were incubated at 300 rpm, 39°C for 4 hours on a heat block. Place on ice after incubation.

[0667] AA VX purification

[0668] The in vitro filling reaction was diluted with one volume of PBS and subject to AAVX purification as described in Example 3. The sample was analyzed with qPCR as described in the “Analytical procedures” section below. Results are shown in Figure 31. Figure 31 shows AAV titers determined by qPCR; and fill rates as determined by ELISA and qPCR of chemically defined in vitro filling reactions using optimal combinations of recombinant Rep proteins.

[0669] Analytical procedures qPCR qPCR analysis was performed essentially as described in Example 1 with the major difference that the primer amplicon was located in the transgene region of the AAV genome. Data shown in Figure 31 . SDS-PAGE

[0670] Expression and purification MBP-Rep78 was assessed using SDS-PAGE and Western blotting. Data shown in Figure 29 and Figure 30, respectively.

[0671] Conclusions

[0672] This works describes:

[0673] 1 . The first demonstration of chemically defined in vitro capsid filling demonstrating maximum fill rates (-100%) of transgene in AAV5

[0674] 2. The novel method of in vitro filling of AAV using a non-liberated Rep fusion protein, MBP-Rep78

[0675] 3. Versatility of fusion proteins (SUMO and MBP) that enhance solubility of Rep proteins while retaining in vitro activity.

[0676] 4. How different combinations of rep proteins lead to different capsid fill rates. In this example, the combination of exclusively using Rep40 and Rep78 showed maximum capsid filling rates.

[0677] Example 9 Truncation Analysis of in vitro filled capsids: proof of genome preservation Introduction

[0678] Adeno-associated viruses (AAVs) exhibit significant variability in composition, payload, and infectivity. Truncation notably diminishes payload expression, thereby reducing treatment efficacy and overall therapeutic potency, which can compromise patient safety. To date, studies specifically targeting fully filled particles are limited, although extensive research has been conducted on quantifying the ratio of empty, partially filled, and fully filled AAVs (Ebberink et al. 2022, Wagner et al. 2024, Wu et al. 2022, and McColl-Carboni et al. 2025). Herein, the inventors delineate a method to quantify the ratio of full, partially filled, and empty particles, and present data illustrating this ratio.

[0679] This data commences with purified chemically defined in vitro filled AAVs, the production of which is described in Example 8.

[0680] For this data, truncation was determined using mass photometry. This technique analyzes biomolecules at the single-particle level in their native state, obviating the need for labels. Mass photometry (MP) experiments provide insights into the molecular weight of all components present in a sample. In MP, a single molecule that comes in contact with a measurement surface (e.g. a coverslip) is exposed to a beam of light and produces a small but measurable light scattering signal, which is directly proportional to the molecule’s mass. The greater the mass of the molecule, the more intense the signal. Due to the differences in mass between empty, partially filled, and fully filled particles, it is feasible to distinguish between these particle types. By quantifying the counts of each particle type, the ratio between them can be accurately determined. Here the inventors show, using in vitro filling and analysing by mass photometry, significantly higher AAV particles containing the complete nucleic acid pay load when compared to the current state of the art AAV production methods. Materials

[0681] Equipment

[0682] Materials

[0683] Methods

[0684] Preparative procedures

[0685] Components generation As per Example 8 and outlined below.

[0686] Empty capsid generation

[0687] AAV5 plasmid and empty capsid generation was performed as described in Example 4. Purification techniques as adopted in Example 3.

[0688] HEK-AA V reference material generation The H EK-fi lied AAV material was generated using the previously stated AAV-MAX Helper-free AAV Production System user guide (Publication number MAN0019619) from Gibco. Purification techniques as adopted in Example 3. Rep Protein generation

[0689] Rep sequence design

[0690] Four Rep proteins are encoded in a single Rep gene in the wtAAV genome. The different proteins are expressed from a combination of alternate splicing and leaky start codons. In order to separately express Rep40, Rep52, Rep 68, Rep78 as four separate proteins, the amino acid sequences had to be extrapolated from Rep. Once determined, each Rep protein sequence was combined with an N-terminal cleavable SUMO fusion domain containing a purification tag, improving solubility, and a C-terminal ‘c-tag’ (aa sequence: EPEA) for purification (sequences of SEQ ID NOs: 40-43 listed in ’’Sequence Overview”). Thus four plasmid designs were created, one for each Rep protein. For each Rep protein a new cell bank was created. Methods described in sections below were run four times in parallel, one for each of the four rep proteins.

[0691] Transformation and cell bank

[0692] The codon-optimized synthetic gene sequence of the target gene coding for His6-SUMO-Rep-EPEA was transformed into 25 pL competent BL21 (DE3) strain by adding 0.5 pL 50 ng / pL of target plasmid DNA. The mixture was flicked to thoroughly mix and incubated on ice for 30 min. Cells were then heat shocked for exactly 10 seconds at 42°C and placed on ice for 5 min. 475 pL room temperature SOC medium was added, and the cells was placed at 37°C for 60 min with 250 rpm shaking in a benchtop shaker. 200 pL was then spread onto a selective plate consisting of LB medium containing 50 mg / L kanamycin as selection marker and incubated overnight at 37°C. A scrape of transformed E. coli-cultures from selective plates was used to inoculate 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. The culture was incubated overnight in an incubator with shaking at 250 rpm at 37°C. The following morning, 25 mL autoclaved LB medium supplemented with 50 mg / L kanamycin was inoculated with 250 pL of the overnight culture and placed at 37°C with 250 rpm shaking in an incubator. When ODeoo reached 1-2, sterilized glycerol was added to a final concentration of 15% and shaken until homogenous. The medium was aliquoted in 10x1 mL labelled vials and stored at -70°C as MCB. The presence of the full-length Rep fusion protein was confirmed by SDS-PAGE.

[0693] Production

[0694] To scale up expression, a scrape from 1 MCB vial of each of the four constructs was used to inoculate a 15 mL falcon tube with 5 mL sterile filtered LB medium containing 50 mg / L kanamycin. Cells were cultured overnight in a shaking incubator at 37.0°C with 250 rpm shaking. The following day, 4 x 2L sterile baffled shake flasks with 500 mL 2xYT medium supplemented with 50 mg / L kanamycin was inoculated with 5 mL of the overnight culture. Flasks were placed in a shaking incubator at 250 rpm at 37.0°C for approximately 3 hours. When the ODeoo reached -0.8-1 .2, cells were induced by adding IPTG to a final concentration of 1 mM. The shake flasks were incubated overnight at 18°C in the same incubator and same shaking. The following morning, cells were pelleted by centrifugation at 15,000g for 15 minutes at 4°C using a fixed-angel rotor. Supernatants were discarded and pellets were stored at -20°C until further use.

[0695] Cell lysis and purification

[0696] Cells were resuspended in 15 mL lysis buffer (20 mM Tris HCI pH 8.0, complete protease inhibitor, 5 mM DTT, 7 U / ml Denarase) in a 50 mL falcon tube and lysed by sonication for 10 minutes with pulses of 2 seconds on and 4 seconds off at an amplitude of 70%, keeping the sample in an ice bath to prevent heating. Insoluble material was removed by centrifugation at 15,000g for 15 minutes using a fixed angle rotor at 4°C. The supernatant was sterile filtered through a 0.2 pm filter and loaded onto a CaptureSelect C-tagXL MiniChrom column equilibrated in equilibration buffer (100 mM Tris HCI pH 8.0). The column was washed with a wash buffer (50 mM Tris HCI pH 8.0, 150 mM NaCI) and the protein of interest was eluted using an elution buffer (50 mM Tris HCI pH 8.0, 2 M MgCh). SDS-PAGE and Western blotting (data not shown) using an anti-AAV2 Replicase mouse monoclonal antibody was performed to positively identify the presence, purity, and identity of the proteins of interest.

[0697] Dialysis and proteolytic activation

[0698] Purified samples were dialyzed against PBS pH 7.4 containing 10% glycerol using a 10 kDa cutoff. Dialysis was performed at 5°C while slowly stirring. After at least 3 hours, the dialysis buffer was exchanged with fresh dialysis buffer and continued overnight. The following morning, the samples were centrifuged at 15,000g for 10 min at 5°C in a benchtop centrifuge. SUMO protease cleavage was carried out under these conditions: all target protein was adjusted to 10 pM in a 500 pL setup and incubated for 16 hours at 5°C and at room temperature with 0.1 U / pL SUMO protease and 1 mM DTT. Cleavage was verified by SDS-PAGE and Western blot (data not shown) using an anti-AAV2 Replicase mouse monoclonal antibody.

[0699] In vitro filling buffer preparation

[0700] HEPES 30mM, pH 6.0 solution was supplemented with the following:

[0701] Table 31 Base buffer components

[0702] The following working solutions were used:

[0703] • CPK solution (1000|jg / mL): prepared as described in Example 1

[0704] • Clarified lysate as described above. • GFP plasmid (0.9pg / pL)

[0705] • ATP solution (200mM)

[0706] In vitro AA V5 capsid filling

[0707] The following conditions were prepared on ice:

[0708] Table 32. AAV5 in vitro filled particles

[0709] Table 34. Control without pDNA

[0710] Table 36. control without capsids

[0711] Reactions were incubated at 300 rpm, 39°C for 4 hours on a heat block and analyzed with qPCR.

[0712] Cleaning of the glass cover slips

[0713] Glass cover slips were placed in a custom Teflon holder and immersed in a 1 :1 ethanol:acetone solution, followed by sonication for 20 minutes. The cover slips were then subjected to three wash cycles with MilliQ water. Subsequently, the cover slips were immersed in MilliQ water and sonicated for an additional 20 minutes, followed by three more wash cycles with MilliQ water. Finally, the cover slips were immersed in pure ethanol and sonicated for 20 minutes. The cover slips were dried using a nitrogen stream.

[0714] Analytical procedures

[0715] Mass Photometry

[0716] All mass photometry experiments were carried out at room temperature using a OneMP instrument from Refeyn. The steps below summarize the MP measurement and analysis process.

[0717] 1 . Adhere a 6-well silicone gasket to a clean cover glass.

[0718] 2. Add 18 pL of PBS to a new sample well for each measurement.

[0719] 3. Focus the interface between the buffer and the cover glass surface.

[0720] 4. Introduce 2 pL of sample into the same well. 5. Binding to the cover glass surface was imaged and recorded for 2 minutes at a frame rate of 1 kHz.

[0721] Each sample well was used only once. Reproducibility of the mass photometry measurements was validated through independent experiments using the same sample.

[0722] Data processing

[0723] All mass photometry images were processed and analyzed using DiscoverMP (Refeyn). The interference intensities of the vectors were converted to masses through calibration with known mass standards. Gaussian functions were fitted to the individual peaks, with weights assigned to represent the empty, partially filled, and fully filled adeno-associated viruses (AAVs).

[0724] Results and observations

[0725] Upon analyzing the chemically defined in vitro filled AAVs (Figure 32, longdashed line) and the HEK-filled AAVs (Figure 32, dotted line), it becomes evident that the distribution spread is significantly broader for the former. Both AAVs exhibited an empty capsid peak around 2.5 MDa; however, only the chemically defined in vitro filled AAVs reached a molecular weight of up to 3.5 MDa (Figure 32). In contrast, the HEK-filled material did not exceed a molecular weight of 3.1 MDa (Figure 32). Given that the full payload of the plasmid used for packaging was 2,778 base pairs (corresponding to a molecular weight of 0.9 MDa), it appeared that only the chemically defined in vitro filled AAVs were capable of packaging the full pay load.

[0726] The peak with the highest molecular weight for HEK-filled AAVs (Figure 32, 3.1 MDa) did not surpass the 0.9 MDa threshold required for a full genome, suggesting the presence of a separate subpopulation of partially filled AAVs. It is known and previously published that the distributions for partially filled AAVs can be relatively broad (McColl-Carboni et al 2024, Wu et al., 2010).

[0727] These observations supported the hypothesis that the HEK-filled material contained an increased proportion of partially filled AAVs and was unable to package the full payload, whereas the chemically defined in vitro filled material included a population with the full genome encapsulated within the particles.

[0728] Conclusions

[0729] • The chemically defined in vitro filled AAVs contained a significant proportion of the population with full-length genomes (2.8 kbp, corresponding to 0.9 MDa difference from empty capsids).

[0730] • The HEK-filled material exhibited a higher proportion of partially filled AAVs and lacked fully filled particles. Alternatively, the population of fully filled particles may be so small that it fell below the detection threshold for accurate quantification using mass photometry. • In vitro filling for producing AAVs yielded a significantly higher population of AAV particles that contained 100% of the gene of interest (Gol) payload. In contrast, the standard methods currently in use produce AAV populations where no detectable amount of AAVs contained the full genomic payload. This advancement represents a substantial improvement over the existing state of the art, as it enhances the efficiency and precision of gene delivery by ensuring that a much greater proportion of AAV vectors carry the complete intended therapeutic payload.

[0731] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication and patent application was specifically and individually indicated to be incorporated by reference.

[0732] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, and adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known and customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0733] Other embodiments are within the claims.

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[0763] US 6,156,303; Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom WO 00 / 28061 , ADENO-ASSOCIATED VIRUS SEROTYPE 1 NUCLEIC ACID SEQUENCES, VECTORS AND HOST CELLS CONTAINING SAME.

[0764] \NO 98 / 11244; AAV4 VECTOR AND USES THEREOF

[0765] WO 99 / 61601 , AAV5 VECTOR AND USES THEREOF

[0766] Sequence Overview

[0767] SEQ ID NO:1 (VP1 AAV Serotype 2)

[0768] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEA

[0769] DAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVK

[0770] TAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMA

[0771] TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNH

[0772] YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV

[0773] QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNN

[0774] FTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWL

[0775] PGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG

[0776] SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQG

[0777] PIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0778] SEQ ID NO:2 (VP1 AAV Serotype 5)

[0779] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRA

[0780] DEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKI-ADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAK

[0781] TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDN

[0782] NQGADGVGNASGDWHCDSTWMGDRWTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPW

[0783] GYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQ

[0784] LPYWGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFE

[0785] EVPFHSSFAPSQNLFKUKNPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGVVNL

[0786] GSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEG

[0787] NMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPET

[0788] GAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRW NPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0789] SEQ ID NO:3 (VP1 AAV Serotype 6)

[0790] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA

[0791] ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEG

[0792] AKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTM

[0793] ASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASND

[0794] NHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTS

[0795] TVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN

[0796] NFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKN WLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGK

[0797] ESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYL

[0798] QGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEW ELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0799] SEQ ID N0:4 (VP1 AAV Serotype 8)

[0800] MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA

[0801] ADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG

[0802] AKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNT

[0803] MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGAT

[0804] NDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNL

[0805] TSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG

[0806] NNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAK

[0807] NWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGK

[0808] QNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYL

[0809] QGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEW

[0810] ELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0811] SEQ ID NO:5 (VP2 AAV Serotype 2)

[0812] TAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMA

[0813] TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNH

[0814] YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV

[0815] QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNN

[0816] FTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWL

[0817] PGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQG

[0818] SEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQG

[0819] PIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0820] SEQ ID NO:6 (VP3 AAV Serotype 2)

[0821] MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASND

[0822] NHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTS

[0823] TVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG

[0824] NNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRN

[0825] WLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGK

[0826] QGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYL QGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEW

[0827] ELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0828] SEQ ID NO:7 (VP1 AAV Serotype 1)

[0829] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNA

[0830] ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG

[0831] AKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTM

[0832] ASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASND

[0833] NHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTS

[0834] TVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN

[0835] NFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKN

[0836] WLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGK

[0837] ESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYL

[0838] QGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEW ELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0839] SEQ ID NO:8 (VP1 AAV Serotype 3)

[0840] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNE

[0841] ADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAA

[0842] KTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTM

[0843] ASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDN

[0844] HYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTST

[0845] VQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN

[0846] NFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQAR

[0847] NWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIF

[0848] GKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDV

[0849] YLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEI

[0850] EWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0851] SEQ ID NO:9 (VP1 AAV Serotype 7)

[0852] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNA

[0853] ADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG

[0854] AKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSSVGSGT

[0855] VAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTN

[0856] DNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLT

[0857] STIQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTG NNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQA

[0858] KNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIF

[0859] GKTGATNKTTLENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQWNNQGALPGMVWQNRDVY LQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL

[0860] SEQ ID NO:10 (VP1 AAV Serotype 9)

[0861] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNA

[0862] ADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAA

[0863] KTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMAS

[0864] GGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDN

[0865] AYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTST

[0866] VQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNN

[0867] FQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP

[0868] GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQG

[0869] TGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGVWQNQGILPGMVWQDRDVYLQ

[0870] GPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0871] SEQ ID NO:11 (VP2 AAV Serotype 1)

[0872] TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMAS

[0873] GGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNH

[0874] YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTV

[0875] QVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNF

[0876] TFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWL

[0877] PGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESA

[0878] GASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQG PIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0879] SEQ ID NO:12 (VP2 AAV Serotype 3b)

[0880] TAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMA

[0881] SGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNH

[0882] YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTV

[0883] QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNN

[0884] FQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARN WLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGK

[0885] EGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYL

[0886] QGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEW

[0887] ELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0888] SEQ ID N0:13 (VP2 AAV Serotype 5)

[0889] TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDN

[0890] NQGADGVGNASGDWHCDSTWMGDRWTKSTRTVWLPSYNNHQYREIKSGSVDGSNANAYFGYSTPW

[0891] GYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQ

[0892] LPYWGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFE

[0893] EVPFHSSFAPSQNLFKUKNPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGVVNL

[0894] GSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEG

[0895] NMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPET

[0896] GAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRW

[0897] NPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0898] SEQ ID NO:14 (VP2 AAV Serotype 6)

[0899] TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMAS

[0900] GGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNH

[0901] YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTV

[0902] QVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNF

[0903] TFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWL

[0904] PGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESA

[0905] GASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQG

[0906] PIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQ

[0907] KENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0908] SEQ ID NO:15 (VP2 AAV Serotype 7)

[0909] TAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSSVGSGTVA

[0910] AGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTNDN

[0911] TYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTI

[0912] QVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNF

[0913] EFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYI-ARTQSNPGGTAGNRELQFYQGGPSTMAEQAKN

[0914] WLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGK

[0915] TGATNKTTLENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQWNNQGALPGMVWQNRDVYLQ GPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL

[0916] SEQ ID N0:16 (VP2 AAV Serotype 8)

[0917] TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMA

[0918] AGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATND

[0919] NTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTS

[0920] TIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN

[0921] NFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKN

[0922] WLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQ

[0923] NAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQ

[0924] GPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0925] SEQ ID NO:17 (VP2 AAV Serotype 9)

[0926] TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMAS

[0927] GGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDN

[0928] AYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTST

[0929] VQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNN

[0930] FQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP

[0931] GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQG

[0932] TGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQ GPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0933] SEQ ID NO:18 (VP3 AAV Serotype 1)

[0934] MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASN

[0935] DNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLT

[0936] STVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG

[0937] NNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPK

[0938] NWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFG

[0939] KESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVY

[0940] LQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIE WELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0941] SEQ ID NO:19 (VP3 AAV Serotype 3b) MASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASND

[0942] NHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTS

[0943] TVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG

[0944] NNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQA

[0945] RNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIF

[0946] GKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDV YLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEI EWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0947] SEQ ID NO:20 (VP3 AAV Serotype 5)

[0948] MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRWTKSTRTVWLPSYNNHQYREIKSGSVDG

[0949] SNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANN

[0950] LTSTVQVFTDDDYQLPYWGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKM

[0951] LRTGNNFEFTYNFEEVPFHSSFAPSQNLFKI_ANPLVDQYLYRFVSTNNTGGVQFNKNI_AGRYANTYKNW

[0952] FPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNS

[0953] QPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMER DVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVE MEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0954] SEQ ID NO:21 (VP3 AAV Serotype 6)

[0955] MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASN

[0956] DNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLT

[0957] STVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG

[0958] NNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPK

[0959] NWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFG

[0960] KESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVY

[0961] LQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIE WELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0962] SEQ ID NO:22 (VP3 AAV Serotype 7)

[0963] VAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTN

[0964] DNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLT

[0965] STIQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTG

[0966] NNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQA

[0967] KNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIF

[0968] GKTGATNKTTLENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQWNNQGALPGMVWQNRDVY LQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIE

[0969] WELQKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL

[0970] SEQ ID NO:23 (VP3 AAV Serotype 8)

[0971] MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGAT

[0972] NDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNL

[0973] TSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG

[0974] NNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAK

[0975] NWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGK

[0976] QNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYL

[0977] QGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEW

[0978] ELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0979] SEQ ID NO:24 (VP3 AAV Serotype 9)

[0980] MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSS

[0981] NDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL

[0982] TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0983] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGR

[0984] NYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFG

[0985] KQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRD

[0986] VYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVE

[0987] IEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0988] SEQ ID NO:25 (pAAV-RC2 Vector sequence) [131-1996: AAV-2 Rep gene, 2013-4220: AAV-2 Cap gene

[0989] 5292-6152: Ampicillin Resistance (bla) gene]

[0990] GCGCGCCGATATCGTTAACGCCCCGCGCCGGCCGCTCTAGAACTAGTGGATCCCCCGGAAGATCA

[0991] GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGATCTGCGCAGCCGCCATG

[0992] CCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTG

[0993] ACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGA

[0994] ATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGC

[0995] GCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCC

[0996] ACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGAT

[0997] TCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTC

[0998] ACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTAC

[0999] TTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCT

[1000] GTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGG AGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAG

[1001] GTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGG

[1002] AGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTT

[1003] GGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCC

[1004] CGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATG

[1005] CGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTG

[1006] GGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGT

[1007] GCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGA

[1008] GGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGC

[1009] GCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACA

[1010] CCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACC

[1011] GGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGT

[1012] CAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAG

[1013] GGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGA

[1014] GTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAA

[1015] CAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAAT

[1016] CAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATC

[1017] TCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGG

[1018] TGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATA

[1019] AATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAA

[1020] GGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAG

[1021] GACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAG

[1022] GGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGC

[1023] TCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTA

[1024] AAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTG

[1025] AACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACT

[1026] CTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGA

[1027] TTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCA

[1028] GCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAAT

[1029] AACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGC

[1030] GACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAA

[1031] CAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGT

[1032] ATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAAC

[1033] TGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGA

[1034] ATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTA

[1035] CCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTT CATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTT

[1036] TACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTT

[1037] TGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTC

[1038] ATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGC

[1039] TTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCT

[1040] GTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGG

[1041] AGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCA

[1042] CAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAG

[1043] AAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCG

[1044] TGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCG

[1045] CAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGG

[1046] GGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATT

[1047] CGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACC

[1048] ACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGA

[1049] TCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTA

[1050] CAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATT

[1051] GGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTT

[1052] GAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGG

[1053] GTTAATCATTAACTACAGCCCGGGCGTTTAAACAGCGGGCGGAGGGGTGGAGTCGTGACGTGAATT

[1054] ACGTCATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACC

[1055] ATGTGGTCTCGCTGGGGGGGGGGGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGT

[1056] TTGAACGAGCGCTGGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCG

[1057] TTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCG

[1058] CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAAATTGTAAGCGTTAATATTT

[1059] TGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTTAACCAATAGGCCGAAATCGGCAAA

[1060] ATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCC

[1061] ACTATTAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTA

[1062] CGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTA

[1063] AAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAG

[1064] AAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC

[1065] ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCG

[1066] GAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT

[1067] AAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT

[1068] TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA

[1069] GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT

[1070] TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCAT AACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAAC CGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTA TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAG TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCG GTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTT CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT GGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGC CGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA TTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTG TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAA

[1071] SEQ ID NO:26 (pHelper Vector sequence) [1-5336: Adenovirus E2A gene, 5337-8537: Adenovirus E4 gene, 8538-9280: Adenovirus VA gene, 10182-11042: Ampicillin Resistance (bla) gene (complement)] GGTACCCAACTCCATGCTTAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACCAGGAACAGCT CTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCAC TTCTTTTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGGAGACACTTTCAATAAAGGCAAATGTT TTTATTTGTACACTCTCGGGTGATTATTTACCCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAA GGGGTTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCT CCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCAC CATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTG

[1072] CGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGC

[1073] TGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAAC

[1074] GGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCAC

[1075] CGTAGTGGCATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGC

[1076] CTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCG

[1077] GAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGC

[1078] ACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGCGCT

[1079] GCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGA

[1080] CACTTAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTG

[1081] GTGCTTGTAGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACA

[1082] AAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGGTCTTGCATA

[1083] CGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCTTTAGATCGTTATCCACGTG

[1084] GTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGCAGGCT

[1085] CAGCGGGTTTATCACCGTGCTTTCACTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCA

[1086] TACCCCGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCT

[1087] TGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCT

[1088] GTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTTTTTCTTTTT

[1089] GGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGC

[1090] GCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCCTCAGCCGCTTTTTTGGGGGC

[1091] GCGCGGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACGTCGCG

[1092] CCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCT

[1093] CCTATAGGCAGAAAAAGATCATGGAGTCAGTCGAGAAGGAGGACAGCCTAACCGCCCCCTTTGAGT

[1094] TCGCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCACCCCCG

[1095] CTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGGATCGC

[1096] TCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACGACGCAGAGGCAAACGAGGAACAAGTCGGG

[1097] CGGGGGGACCAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCA

[1098] GCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGG

[1099] ATGTCAGCCTTGCCTACGAACGCCACCTGTTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAACG

[1100] GCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCA

[1101] CCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGA

[1102] CAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACCTGATATCGCCTCGCTCGACGAAGTGCCAAA

[1103] AATCTTTGAGGGTCTTGGACGCGACGAGAAACGCGCGGCAAACGCTCTGCAACAAGAAAACAGCGA

[1104] AAATGAAAGTCACTGTGGAGTGCTGGTGGAACTTGAGGGTGACAACGCGCGCCTAGCCGTGCTGAA

[1105] ACGCAGCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTTATGAGCACA

[1106] GTCATGAGCGAGCTGATCGTGCGCCGTGCACGACCCCTGGAGAGGGATGCAAACTTGCAAGAACAA ACCGAGGAGGGCCTACCCGCAGTTGGCGATGAGCAGCTGGCGCGCTGGCTTGAGACGCGCGAGC

[1107] CTGCCGACTTGGAGGAGCGACGCAAGCTAATGATGGCCGCAGTGCTTGTTACCGTGGAGCTTGAGT

[1108] GCATGCAGCGGTTCTTTGCTGACCCGGAGATGCAGCGCAAGCTAGAGGAAACGTTGCACTACACCT

[1109] TTCGCCAGGGCTACGTGCGCCAGGCCTGCAAAATTTCCAACGTGGAGCTCTGCAACCTGGTCTCCT

[1110] ACCTTGGAATTTTGCACGAAAACCGCCTCGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGG

[1111] CGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTGTGCTACACCTGGCAAACGGCCATGG

[1112] GCGTGTGGCAGCAATGCCTGGAGGAGCGCAACCTAAAGGAGCTGCAGAAGCTGCTAAAGCAAAACT

[1113] TGAAGGACCTATGGACGGCCTTCAACGAGCGCTCCGTGGCCGCGCACCTGGCGGACATTATCTTCC

[1114] CCGAACGCCTGCTTAAAACCCTGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAAAA

[1115] CTTTAGGAACTTTATCCTAGAGCGTTCAGGAATTCTGCCCGCCACCTGCTGTGCGCTTCCTAGCGAC

[1116] TTTGTGCCCATTAAGTACCGTGAATGCCCTCCGCCGCTTTGGGGTCACTGCTACCTTCTGCAGCTAG

[1117] CCAACTACCTTGCCTACCACTCCGACATCATGGAAGACGTGAGCGGTGACGGCCTACTGGAGTGTC

[1118] ACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCTGGTCTGCAATTCGCAACTGCTTAGCGAAA

[1119] GTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGT

[1120] TGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGC

[1121] CCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGCTTACCGCCTGCGTCAT

[1122] TACCCAGGGCCACATCCTTGGCCAATTGCAAGCCATCAACAAAGCCCGCCAAGAGTTTCTGCTACGA

[1123] AAGGGACGGGGGGTTTACCTGGACCCCCAGTCCGGCGAGGAGCTCAACCCAATCCCCCCGCCGCC

[1124] GCAGCCCTATCAGCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCAAAAAGAAGCTGCAGCTG

[1125] CCGCCGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGA

[1126] GGAGGAGGAGATGATGGAAGACTGGGACAGCCTAGACGAAGCTTCCGAGGCCGAAGAGGTGTCAG

[1127] ACGAAACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATTGGCAACCGTTCCCA

[1128] GCATCGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCTGTTCGCCGACCCAACCGTAGAT

[1129] GGGACACCACTGGAACCAGGGCCGGTAAGTCTAAGCAGCCGCCGCCGTTAGCCCAAGAGCAACAA

[1130] CAGCGCCAAGGCTACCGCTCGTGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGCAAGACTGT

[1131] GGGGGCAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAAC

[1132] ATCCTGCATTACTACCGTCATCTCTACAGCCCCTACTGCACCGGCGGCAGCGGCAGCGGCAGCAAC

[1133] AGCAGCGGTCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCCAC

[1134] AGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGTATCGACCCGC

[1135] GAGCTTAGAAATAGGATTTTTCCCACTCTGTATGCTATATTTCAACAAAGCAGGGGCCAAGAACAAGA

[1136] GCTGAAAATAAAAAACAGGTCTCTGCGCTCCCTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGAT

[1137] CAGCTTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGCAAATACTGCGCGCTGACTCTTAAG

[1138] GACTAGTTTCGCGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGG

[1139] CGCCAGCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGTGGAGTTACC

[1140] AGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTACATGAGCG

[1141] CGGGACCCCACATGATATCCCGGGTCAACGGAATCCGCGCCCACCGAAACCGAATTCTCCTCGAAC AGGCGGCTATTACCACCACACCTCGTAATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTA

[1142] CCAGGAAAGTCCCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGAC

[1143] TAACTCAGGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCGTTTTAGGG

[1144] CGGAGTAACTTGCATGTATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGTATCGTGGGAAAAC

[1145] GGAAGTGAAGATTTGAGGAAGTTGTGGGTTTTTTGGCTTTCGTTTCTGGGCGTAGGTTCGCGTGCGG

[1146] TTTTCTGGGTGTTTTTTGTGGACTTTAACCGTTACGTCATTTTTTAGTCCTATATATACTCGCTCTGTA

[1147] CTTGGCCCTTTTTACACTGTGACTGATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTT

[1148] TTTACTGGTAAGGCTGACTGTTATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGA

[1149] GGGTGCTATTTTGCCTAGGCAGGAGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTAATTTTGT

[1150] TATACCTCCTATGGGGGCTGTAATGTTGTCTCTACGCCTGCGGGTATGTATTCCCCCGGGCTATTTC

[1151] GGTCGCTTTTTAGCACTGACCGATGTTAACCAACCTGATGTGTTTACCGAGTCTTACATTATGACTCC

[1152] GGACATGACCGAGGAACTGTCGGTGGTGCTTTTTAATCACGGTGACCAGTTTTTTTACGGTCACGCC

[1153] GGCATGGCCGTAGTCCGTCTTATGCTTATAAGGGTTGTTTTTCCTGTTGTAAGACAGGCTTCTAATGT

[1154] TTAAATGTTTTTTTTTTTGTTATTTTATTTTGTGTTTAATGCAGGAACCCGCAGACATGTTTGAGAGAAA

[1155] AATGGTGTCTTTTTCTGTGGTGGTTCCGGAACTTACCTGCCTTTATCTGCATGAGCATGACTACGATG

[1156] TGCTTGCTTTTTTGCGCGAGGCTTTGCCTGATTTTTTGAGCAGCACCTTGCATTTTATATCGCCGCCC

[1157] ATGCAACAAGCTTACATAGGGGCTACGCTGGTTAGCATAGCTCCGAGTATGCGTGTCATAATCAGTG

[1158] TGGGTTCTTTTGTCATGGTTCCTGGCGGGGAAGTGGCCGCGCTGGTCCGTGCAGACCTGCACGATT

[1159] ATGTTCAGCTGGCCCTGCGAAGGGACCTACGGGATCGCGGTATTTTTGTTAATGTTCCGCTTTTGAA

[1160] TCTTATACAGGTCTGTGAGGAACCTGAATTTTTGCAATCATGATTCGCTGCTTGAGGCTGAAGGTGG

[1161] AGGGCGCTCTGGAGCAGATTTTTACAATGGCCGGACTTAATATTCGGGATTTGCTTAGAGACATATT

[1162] GATAAGGTGGCGAGATGAAAATTATTTGGGCATGGTTGAAGGTGCTGGAATGTTTATAGAGGAGATT

[1163] CACCCTGAAGGGTTTAGCCTTTACGTCCACTTGGACGTGAGGGCAGTTTGCCTTTTGGAAGCCATTG

[1164] TGCAACATCTTACAAATGCCATTATCTGTTCTTTGGCTGTAGAGTTTGACCACGCCACCGGAGGGGA

[1165] GCGCGTTCACTTAATAGATCTTCATTTTGAGGTTTTGGATAATCTTTTGGAATAAAAAAAAAAAAACAT

[1166] GGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGACTCGCAGAACGAATGTGTAGGTTGGCTGGG

[1167] TGTGGCTTATTCTGCGGTGGTGGATGTTATCAGGGCAGCGGCGCATGAAGGAGTTTACATAGAACC

[1168] CGAAGCCAGGGGGCGCCTGGATGCTTTGAGAGAGTGGATATACTACAACTACTACACAGAGCGAGC

[1169] TAAGCGACGAGACCGGAGACGCAGATCTGTTTGTCACGCCCGCACCTGGTTTTGCTTCAGGAAATAT

[1170] GACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACT

[1171] CCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCAT

[1172] CCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTTCCCTGCA

[1173] GTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTTCTGACGCGGGAGGAGC

[1174] TTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCAT

[1175] GATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGC

[1176] ATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAG AGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCAGCG

[1177] TGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTGGT

[1178] CCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGC

[1179] TGCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTC

[1180] ATGCTGCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAG

[1181] CGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTAT

[1182] GACTCTACCCCCATGTAGGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAGTTGGACAG

[1183] CAGCCTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTGCCCGGGGAGTTTATTAAT

[1184] ATCACTGATGAGCGTTTGGCTCGACAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTA

[1185] CCCATGATATGATGCTTTTTAAGGCCAGCCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGG

[1186] GAGGTGGCAGGTTGAATACTAGGGTTCTGTGAGTTTGATTAAGGTACGGTGATCAATATAAGCTATG

[1187] TGGTGGTGGGGCTATACTACTGAATGAAAAATGACTTGAAATTTTCTGCAATTGAAAAATAAACACGT

[1188] TGAAACATAACATGCAACAGGTTCACGATTCTTTATTCCTGGGCAATGTAGGAGAAGGTGTAAGAGTT

[1189] GGTAGCAAAAGTTTCAGTGGTGTATTTTCCACTTTCCCAGGACCATGTAAAAGACATAGAGTAAGTGC

[1190] TTACCTCGCTAGTTTCTGTGGATTCACTAGAATCGATGTAGGATGTTGCCCCTCCTGACGCGGTAGG

[1191] AGAAGGGGAGGGTGCCCTGCATGTCTGCCGCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGC

[1192] GCATCTGCCGCAGCACCGGATGCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGG

[1193] AATTTGCAAGCGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCC

[1194] GAGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTACAGGGAGCGA

[1195] GCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCGGCCAGTGGCCAAAAAAGC

[1196] TAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCAAAAGGAGCGCTCCCCCGTTGTCTGACGTCGC

[1197] ACACCTGGGTTCGACACGCGGGCGGTAACCGCATGGATCACGGCGGACGGCCGGATCCGGGGTTC

[1198] GAACCCCGGTCGTCCGCCATGATACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCT

[1199] TACAGGCTCTCCTTTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGCGTC

[1200] CCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTTCCGCGCGCC

[1201] TCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGGATTACGTCGACGTTTAAACC

[1202] ATATGATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA

[1203] ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC

[1204] ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA

[1205] CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT

[1206] GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG

[1207] CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT

[1208] TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA

[1209] TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAG

[1210] TTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGA

[1211] AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGA GTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGG CCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAA TTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTA AAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGAT CCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCT

[1212] GGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA ATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATAC ATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACC

[1213] TAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACC AATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCT

[1214] ATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAA AGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGT GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTC

[1215] ACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGATGGATCC

[1216] SEQ ID NO:27 (pAAV-GFP Vector sequence) [1-130: Left ITR, 139-798: CMV Promoter, 806-1298: Human p-globin Intron, 1321-2061 : GFP, 2120-2598: PolyA, 2638-2778: Right ITR, 3695-4555: Ampicillin Resistance]

[1217] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCG CCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT GCGGCCGCACGCGTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT

[1218] TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTC CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATC

[1219] AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGG

[1220] AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCA

[1221] AATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT

[1222] CGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCG

[1223] CGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTA

[1224] AGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCT

[1225] TATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCA

[1226] CCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTT

[1227] CTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT

[1228] CTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATC

[1229] ATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATC

[1230] ACTTTGGCAAAGAATTGGGATTCGAACATCGATTGAATTCTGAATGGTGAGCAAGGGCGAGGAGCTG

[1231] TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGT

[1232] GTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG

[1233] GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGC

[1234] CGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG

[1235] GAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG

[1236] CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGG

[1237] GGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG

[1238] GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT

[1239] ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC

[1240] CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC

[1241] GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTACTCAGATCTCGAGCTCAAGTAGGGA

[1242] TCCTCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCAGCGCTGCTCGAGAGATCTACGGGTGGCAT

[1243] CCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTT

[1244] GTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAG

[1245] GGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGG

[1246] AACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGA

[1247] TTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTT

[1248] GTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTG

[1249] ATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCC

[1250] TTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGC

[1251] CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGG

[1252] GCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCG

[1253] GTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCC CTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCA

[1254] GCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG

[1255] TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA

[1256] AAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA

[1257] CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG

[1258] GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAA

[1259] CAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCT

[1260] GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGG

[1261] GCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG

[1262] AGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGG

[1263] TTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC

[1264] CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATG

[1265] CTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT

[1266] GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATC

[1267] AGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG

[1268] CCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTA

[1269] TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC

[1270] ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC

[1271] ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTT

[1272] TTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT

[1273] ACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACT

[1274] GGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG

[1275] GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC

[1276] GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT

[1277] ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCAC

[1278] TGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTT

[1279] TTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT

[1280] TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGC

[1281] GCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGA

[1282] GCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA

[1283] GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAA

[1284] TCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA

[1285] GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

[1286] GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG

[1287] GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT

[1288] TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA TTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGG

[1289] TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT

[1290] SEQ ID NO:28 (pAAV-RC5 Vector sequence) [121-1986:AAV-2 Rep gene, 2013-4187:AAV-5 Cap gene, 5268-6128:Ampicillin Resistance (bla) gene]

[1291] ATCGTTAACGCCCCGCGCCGGCCGCTCTAGAACTAGTGGATCCCCCGGAAGATCAGAAGTTCCTAT

[1292] TCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGATCTGCGCAGCCGCCATGCCGGGGTTTT

[1293] ACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGT

[1294] GAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGA

[1295] GCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGA

[1296] GTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGT

[1297] GCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAA

[1298] CTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCA

[1299] GAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCA

[1300] AAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCT

[1301] CACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACA

[1302] AAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGA

[1303] GCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGG

[1304] CCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGC

[1305] GGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGG

[1306] ACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCC

[1307] GTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCA

[1308] ACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAAC

[1309] TGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGG

[1310] AAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGA

[1311] CCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACAT

[1312] GTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTT

[1313] CAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGAC

[1314] TTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGA

[1315] GCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTT

[1316] GCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGT

[1317] TCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATT

[1318] CAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCC

[1319] GTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAG

[1320] ACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATT

[1321] TGTAAATAAATTTAGTAGTCATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAA GGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAA

[1322] GATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGTCTCGATCGA

[1323] GGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGTACAACGAGCAGCT

[1324] TGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTCG

[1325] CCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCG

[1326] AACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACT

[1327] TTCCAAAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAG

[1328] CTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTGATA

[1329] CAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGAGTGGGCAAT

[1330] GCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGA

[1331] ACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGA

[1332] AGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACA

[1333] GCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGGTCCC

[1334] TCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGC

[1335] CAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGG

[1336] CAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTA

[1337] CGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTA

[1338] CTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCC

[1339] TTCCACTCCAGCTTCGCTCCCAGTCAGAACCTGTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACT

[1340] TGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGAT

[1341] ACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGC

[1342] TCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGC

[1343] GAGTTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGC

[1344] CCTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGA

[1345] GGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCG

[1346] GCGGGCAGATGGCCACCAACAACCAGAGCTCCACCACTGCCCCCGCGACCGGCACGTACAACCTC

[1347] CAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGACGTGTACCTCCAAGGACCCATCTGGGC

[1348] CAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACA

[1349] CCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGT

[1350] GCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGC

[1351] TCAAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCA

[1352] GTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATA

[1353] CCTTACCCGACCCCTTTAACCCATTCATGTCGCATACCCTCAATAAACCGGTTAATTCGTGTCAGTTG

[1354] AACTTTGGTCTCATGTCGTTATTATCTTATCTGGTCACCAGATCCCCGTAGATAAGTAGCATGGCGGG

[1355] TTAATCATTAACTACAGCCCGGGCGTTTAAACAGCGGGCGGAGGGGTGGAGTCGTGACGTGAATTA

[1356] CGTCATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCA TGTGGTCTCGCTGGGGGGGGGGGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTT

[1357] TGAACGAGCGCTGGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGT

[1358] TACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCG

[1359] CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAAATTGTAAGCGTTAATATTT

[1360] TGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTTAACCAATAGGCCGAAATCGGCAAA

[1361] ATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCC

[1362] ACTATTAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTA

[1363] CGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTA

[1364] AAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAG

[1365] AAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC

[1366] ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCG

[1367] GAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT

[1368] AAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT

[1369] TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA

[1370] GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT

[1371] TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC

[1372] CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT

[1373] ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCAT

[1374] AACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAAC

[1375] CGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA

[1376] GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTA

[1377] TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAG

[1378] TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCG

[1379] GTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG

[1380] TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG

[1381] CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC

[1382] TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC

[1383] GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

[1384] TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG

[1385] ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTT

[1386] CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC

[1387] TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG

[1388] ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT

[1389] TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC

[1390] CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG

[1391] GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT

[1392] TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT

[1393] GGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG

[1394] CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGC

[1395] CGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA

[1396] TTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTG

[1397] TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCG CCGAT

[1398] SEQ ID NO:29 (Smt3-Rep40)

[1399] MHHHHHHGSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKE

[1400] MDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGMELVGWLVDKGITSEKQWIQEDQASYISFNAA

[1401] SNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKR

[1402] NTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKWESAKAILGGS

[1403] KVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVK

[1404] DFFRWAKDHWEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRLARGHSL EPEA

[1405] SEQ ID NO:30 (Smt3-Rep52)

[1406] MHHHHHHGSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKE

[1407] MDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGMELVGWLVDKGITSEKQWIQEDQASYISFNAA

[1408] SNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKR

[1409] NTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKWESAKAILGGS

[1410] KVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVK

[1411] DFFRWAKDHWEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSR

[1412] HVGMNLMLFPCRQCERMNQNSNICFTHGQKDCLECFPVSESQPVSWKKAYQKLCYIHHIMGKVPDACT ACDLVNVDLDDCIFEQEPEA

[1413] SEQ ID N0:31 (Smt3-Rep68)

[1414] MHHHHHHGSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKE

[1415] MDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGMPGFYEIVIKVPSDLDGHLPGISDSFVNWVAE

[1416] KEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVK

[1417] SMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKWDECYIPNYLLPKTQPELQWAWTN

[1418] MEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEK

[1419] QWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYA

[1420] ASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGK MTAKWESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELT

[1421] RRLDHDFGKVTKQEVKDFFRWAKDHWEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSD

[1422] AEASINYADRLARGHSLEPEA

[1423] SEQ ID NO:32 (Smt3-Rep78)

[1424] MHHHHHHGSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLME...

Claims

Claims1 . A method of in vitro assembling parvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby assembling parvovirus capsids and providing assembled parvovirus capsids.

2. A method of in vitro filling parvovirus capsids, comprising the steps of: a. providing: i. one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro; ii. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain in vitro; andHi. a Rep protein in vitro; b. combining the one or more populations of assembled parvovirus capsids, the nucleic acid molecule, and the Rep protein in vitro under filling conditions which allow filling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids and providing filled parvovirus capsids.

3. A method of in vitro filling parvovirus capsids, comprising the steps of: a. providing: i. a population of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro; ii. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain in vitro;Hi. a Rep proteins in vitro; and iv. a reaction mixture in vitro; b. combining the population of assembled parvovirus capsids, the nucleic acid molecule, the Rep protein and the reaction mixture in vitro under filling conditions which allow filling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids and providing filled parvovirus capsids.

4. A method of in vitro manufacturing filled parvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby providing one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro d. providing: i. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain in vitro; and ii. a Rep protein in vitro; e. combining the population of assembled parvovirus capsids obtained in c., the nucleic acid molecule and the Rep protein in vitro under filling conditions which allow filling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids, thereby manufacturing filled parvovirus capsids.

5. A method of in vitro manufacturing filled parvovirus capsids, comprising the steps of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby providing one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins in vitro d. providing: i. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain in vitro; ii. a Rep protein in vitro; andiii. a reaction mixture in vitro e. combining the population of assembled parvovirus capsids obtained in c., the nucleic acid molecule, the Rep protein and the reaction mixture in vitro under filling conditions which allow filling of the assembled capsids with the nucleic acid molecule by the Rep protein, thereby filling parvovirus capsids, thereby manufacturing filled parvovirus capsids.

6. A method of in vitro controlling the stoichiometry of parvovirus viral capsid proteins in assembled parvovirus capsids, comprising the step of: a. providing one or more populations of inhibited parvovirus viral capsid proteins in vitro, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by an assembly inhibitor wherein the one or more populations of inhibited parvovirus viral capsid proteins are provided at a defined stoichiometry; b. combining the inhibited parvovirus viral capsid proteins in vitro; and c. contacting an assembly activator and the inhibited parvovirus viral capsid proteins of step (b) in vitro, wherein the assembly activator disinhibits the assembly inhibitor, thereby allowing the assembly of parvovirus capsids comprising parvovirus viral capsid proteins into assembled parvovirus capsids, thereby assembling parvovirus capsids and providing assembled parvovirus capsids with a defined stoichiometry of parvovirus viral capsid proteins.

7. The method according to any one of the preceding claims, wherein the viral capsid proteins are from a Densovirinae, Hamaparvovirinae, or Parvovirinae.

8. The method according to any one of the preceding claims, wherein the viral capsid proteins are from a Parvovirinae selected from the group consisting of an Amdoparvovirus, Arti parvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetraparvovirus, preferably Dependoparvovirus.

9. The method according to any one of the preceding claims, wherein the viral capsid proteins are from an adeno-associated virus (AAV).

10. The method according to any one of the preceding claims, wherein the viral capsid proteins are synthetic viral capsid proteins and / or recombinant viral capsid proteins.11 . The method according to any one of the preceding claims, wherein the viral capsid proteins are selected from the group consisting of viral protein 1 (VP1), viral protein 2 (VP2), and / or viral protein (VP3).

12. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP1.

13. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP2.

14. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP3.

15. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP1 and VP2.

16. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP1 and VP3.

17. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP2 and VP3.

18. The method according to any one of the preceding claims, wherein the viral capsid proteins are VP1 , VP2, and VP3.

19. The method according to any one of the preceding claims, wherein the VP1 , VP2 and / or VP3 are synthetic VP1 , VP2 and / or VP3 and / or recombinant VP1 , VP2 and / or VP3.

20. The method according to any one of the preceding claims, wherein the VP1 , VP2 and / or VP3 are AAV VP1 , AAV VP2 and / or AAV VP3.21 . The method according to any one of the preceding claims, wherein the AAV is an AAV type 1 , AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, rh 10 AAV, rh.39 AAV, or any other AAV now known and later discovered.

22. The method according to any one of the preceding claims, wherein the VP1 is a VP1 from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

23. The method according to any one of the preceding claims, wherein the VP2 is a VP2 from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh7424. The method according to any one of the preceding claims, wherein the VP3 is a VP3 from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

25. The method according to any one of the preceding claims, wherein the VP1 , VP2, and / or VP3 are from different AAV serotypes.

26. The method according to any one of the preceding claims, wherein the VP1 , VP2, and / or VP3 are from the same AAV serotype.

27. The method according to any one of the preceding claims, wherein the viral capsid proteins, such as VP1 , VP2 and / or VP3, is produced in a eukaryotic microbial system, such as yeast, such a methylotrophic yeast.

28. The method according to any one of the preceding claims, wherein the viral capsid proteins, such as VP1 , VP2 and / or VP3, is produced in an eukaryotic microbial system, such as P. pastoris or S. cerevisiae.

29. The method according to any one of the preceding claims, wherein the viral capsid proteins, such as VP1 , VP2 and / or VP3, is produced in an prokaryotic microbial system, such as E. Coli.

30. The method according to any one of the preceding claims, wherein the viral capsid proteins, such as VP1 , VP2 and / or VP3, is produced a mammalian cell system, such as HEK, CHO, HeLa or Vero cells, an insect cell system, such as Sf9, Sf21 or High Five cells, a plant system, such as tobacco BY, an algae system, a metazoan cell system, a zebrafish and / or cell-free expression systems.31 . The method according to any one of the preceding claims, wherein the inhibited parvovirus viral capsid proteins are inhibited from assembling by multiple assembly inhibitors, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly inhibitors.

32. The method according to any one of the preceding claims, wherein the assembly inhibitor comprises multiple assembly inhibitors, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly inhibitors.

33. The method according to any one of the preceding claims, wherein the assembly inhibitor is selected from the group consisting of a chemically assembly inhibitor, a steric assembly inhibitor and / or a kinetically assembly inhibitor.

34. The method according to any one of the preceding claims, wherein the assembly inhibitor is selected from the group consisting of inhibitory pH, inhibitory salt concentration, denaturing, precipitation conditions and / or an antibody.

35. The method according to any one of the preceding claims, wherein the assembly inhibitor is selected from the group consisting of a retainment of the viral capsid proteins in a compartment and / or physical blockade, such as a cleavable amino acid sequence attached to the viral capsid proteins.

36. The method according to any one of the preceding claims, wherein the assembly inhibitor is selected from the group consisting of inhibitory temperature and / or inhibitory viral capsid protein concentration.

37. The method according to any one of the preceding claims, wherein the assembly inhibitor is selected from the group consisting of an inhibitory modification to the viral capsid proteins, such as a cleavable amino acid sequence, an inhibitory exogenous molecule, inhibitory pH, inhibitory ionic strength, inhibitory conductivity, time, inhibitory temperature, hydrophobicity, inhibitory viral capsid protein concentration, inhibitory viscosity, denaturation, pressure and radiation, such as light, such as ultraviolet light.

38. The method according to any one of the preceding claims, wherein the assembly inhibitor is an inhibitory modification to the viral capsid proteins.

39. The method according to any one of the preceding claims, wherein the assembly inhibitor is a cleavable amino acid sequence attached to the viral capsid proteins.

40. The method according to any one of the preceding claims, wherein the cleavable amino acid sequence is a SUMO tag and / or a thrombin cleavage site.41 . The method according to any one of the preceding claims, wherein the assembly inhibitor is denaturation of the viral capsid proteins.

42. The method according to any one of the preceding claims, wherein the denaturation is thermally- induced denaturation and / or chemically-induced denaturation.

43. The method according to any one of the preceding claims, wherein the denaturation is caused by pH, sonication, ionic strength, conductivity and / or temperature.

44. The method according to any one of the preceding claims, wherein the denaturation is caused by pH.

45. The method according to any one of the preceding claims, wherein the denaturation is caused by ionic strength and / or conductivity.

46. The method according to any one of the preceding claims, wherein the denaturation is caused by temperature.

47. The method according to any one of the preceding claims, wherein the assembly inhibitor is guanidine, such as 1 to 15 M urea, 1 to 14 M urea, 1 to 13 M urea, 1 to 12 M urea, 1 to 11 M urea, 1 to 10 M urea or 1 to 9 M urea48. The method according to any one of the preceding claims, wherein the assembly inhibitor is urea, such as 1 to 15 M urea, 2 to 14 M urea, 3 to 13 M urea, 4 to 12 M urea, 5 to 11 M urea, 6 to 10 M urea or 7 to 9 M urea.

49. The method according to any one of the preceding claims, wherein the assembly inhibitor is NaCI, such as 1 to 15 M NaCI, 2 to 14 M NaCI, 3 to 13 M NaCI, 4 to 12 M NaCI, 5 to 11 M NaCI, 6 to 10 M NaCI or 7 to 9 M NaCI.

50. The method according to any one of the preceding claims, wherein the assembly inhibitor is KCI, such as 1 to 15 M KCI, 2 to 14 M KCI, 3 to 13 M KCI, 4 to 12 M KCI, 5 to 11 M KCI, 6 to 10 M KCI or 7 to 9 M KCI.51 . The method according to any one of the preceding claims, wherein the inhibitory pH is pH below or equal to 5, such as pH below or equal to 4.5, such as pH below or equal to 4, such as pH below or equal to 3.5, such as pH below or equal to 3, such as pH below or equal to 2.5, such as pH below or equal to 2, such as pH below or equal to 1 .5.

52. The method according to any one of the preceding claims, wherein the inhibitory pH is pH above or equal to 8.5, such as pH above or equal to 9, such as pH above or equal to 9.5, such as pH above or equal to 10, such as pH above or equal to 10.5, such as pH above or equal to 11 , such as pH above or equal to 11 .5, such as pH above or equal to 12, such as pH above or equal to 12.5, such as pH above or equal to 13.

53. The method according to any one of the preceding claims, wherein the inhibitory temperature is a temperature between -196 °C to 0 °C, such as -190 °C to 0 °C, such as -180 °C to 0 °C, such as - 170 °C to 0 °C, such as -160 °C to 0 °C, such as -150 °C to 0 °C, such as -140 °C to 0 °C, such as -130 °C to 0 °C, such as -120 °C to 0 °C, such as -110 °C to 0 °C, such as -100 °C to 0 °C, such as -90 °C to 0 °C, such as -80 °C to 0 °C, such as -70 °C to 0 °C, such as -60 °C to 0 °C, such as -50 °C to 0 °C, such as -40 °C to 0 °C, such as -30 °C to 0 °C, such as -20 °C to 0 °C, such as -10 °C to 0 °C.

54. The method according to any one of the preceding claims, wherein the inhibitory temperature is a temperature between -196 °C to -1 °C, such as -196 °C to -10 °C, such as -196 °C to -20 °C, such as -196 °C to -40 °C, such as -196 °C to -50 °C, such as -196 °C to -60 °C, such as -196 °C to -70 °C °C, such as -196 °C to -80 °C, such as -196 °C to -90 °C, such as -196 °C to -100 °C, such as -196 °C to -110 °C, such as -196 °C to -120 °C °C, such as -196 °C to -130 °C, such as - 196 °C to -140 °C, such as -196 °C to -150 °C, such as -196 °C to -160 °C, such as -196 °C to -170 °C, such as -196 °C to -180 °C, such as -196 °C to -190 °C.

55. The method according to any one of the preceding claims, wherein the inhibitory temperature is a temperature between 40 °C to 280 °C, such as between 40 °C to 250 °C, such as between 40 °C to 220 °C, such as between 40 °C to 210 °C, such as between 40 °C to 200 °C, such as between 40 °C to 190 °C, such as between 40 °C to 180 °C, such as between 40 °C to 170 °C, such as between 40 °C to 160 °C, such as between 50 °C to 160 °C, such as between 60 °C to 160 °C, such as between 70 °C to 160 °C, such as between 80 °C to 160 °C, such as between 80 °C to 150 °C, such as between 80 °C to 140 °C, such as between 90 °C to 140 °C, such as between 90 °C to 140 °C, such as between 100 °C to 140 °C, such as between 100 °C to 130 °C, such as between 110 °C to 130 °C.

56. The method according to any one of the preceding claims, wherein the inhibitory exogenous molecule is selected from the group consisting of an antibody, a nucleic acid molecule, a peptide, a protein, a nanobody, a lipid, a sugar, a carbohydrate, a polymer, a small molecule, and / or an aptamer.

57. The method according to any one of the preceding claims, wherein the inhibitory ionic strength and / inhibitory conductivity is a solution comprising NaCI, KCI and / or urea, such as a solution comprising 1 to 15 M NaCI, KCI and / or urea, 2 to 14 M NaCI, KCI and / or urea, 3 to 13 M NaCI, KCI and / or urea, 4 to 12 M NaCI, KCI and / or urea, 5 to 11 M NaCI, KCI and / or urea, 6 to 10 M NaCI, KCI and / or urea or 7 to 9 M NaCI, KCI and / or urea.

58. The method according to any one of the preceding claims, the step of contacting the assembly activator and the inhibited parvovirus viral capsid proteins of step (b) comprise contacting multiple assembly activators, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly activators.

59. The method according to any one of the preceding claims, wherein the assembly activator comprises multiple assembly activators, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 assembly activators.

60. The method according to any one of the preceding claims, wherein the assembly activator is selected from the group consisting of a chemically assembly activator, a steric assembly activator and / or a kinetically assembly activator61 . The method according to any one of the preceding claims, wherein the assembly activator is selected from the group consisting of activating pH, activating salt concentration, renaturing, solubilization conditions and / or removal of the antibody.

62. The method according to any one of the preceding claims, wherein the assembly activator is selected from the group consisting of a release of the viral capsid proteins from the compartment and / or removal of the physical blockade, such as the cleavable amino acid sequence attached to the viral capsid proteins.

63. The method according to any one of the preceding claims, wherein the assembly activator is selected from the group consisting of activating temperature and / or activating viral capsid protein concentration.

64. The method according to any one of the preceding claims, wherein the assembly activator is selected from the group consisting of a removal of the inhibitory modification to the viral capsidproteins, such as a cleavage of the cleavable amino acid sequence, removal of the inhibitory exogenous molecules, activating pH, activating ionic strength, activating conductivity, activating time, activating temperature, hydrophobicity, activating viral capsid protein concentration, renaturation, activating viscosity, agitation, mixing, pressure and radiation, such as light, such as ultraviolet light.

65. The method according to any one of the preceding claims, wherein the assembly activator is a removal of the inhibitory modification to the viral capsid proteins.

66. The method according to any one of the preceding claims, wherein the assembly activator is a cleavage of the cleavable amino acid sequence attached to the viral capsid proteins.

67. The method according to any one of the preceding claims, wherein the assembly activator is a protease.

68. The method according to any one of the preceding claims, wherein the assembly activator is thrombin and / or a SUMO protease, such as SUMO protease 2.

69. The method according to any one of the preceding claims, wherein the cleavable amino acid sequence is a SUMO tag and / or a thrombin cleavage site, and wherein the assembly activator is thrombin and / or a SUMO protease, such as SUMO protease 2.

70. The method according to any one of the preceding claims, wherein the assembly activator is renaturation.71 . The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, such as 1 to 500 mM MES pH 6.0, such as 1 to 450 mM MES pH 6.0, such as 1 to 400 mM MES pH 6.0, such as 1 to 350 mM MES pH 6.0, such as 1 to 300 mM MES pH 6.0, such as 1 to 250 mM MES pH 6.0, such as 1 to 200 mM MES pH 6.0, such as 1 to 150 mM MES pH 6.0, such as 1 to 100 mM MES pH 6.0, such as 1 to 90 mM MES pH 6.0, such as 1 to 80 mM MES pH 6.0, such as 1 to 70 mM MES pH 6.0, such as 10 to 70 mM MES pH 6.0, such as 20 to 70 mM MES pH 6.0, such as 30 to 70 mM MES pH 6.0, such as 40 to 60 mM MES pH 6.0, such as 45 to 55 mM MES pH 6.0.

72. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising NaCI, such as 100 to 1000 mM NaCI, such as 100 to 900 mM NaCI, such as 100 to 800 mM NaCI, such as 100 to 700 mM NaCI, such as 100 to 600 mM NaCI, such as 200 to600 mM NaCI, such as 300 to 600 mM NaCI, such as 400 to 600 mM NaCI, such as 450 to 550 mM NaCI.

73. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising NaCI, such as 100 to 400 mM NaCI, such as 150 to 400 mM NaCI, such as 200 to 350 mM NaCI, such as 200 to 300 mM NaCI, such as 200 to 290 mM NaCI, such as 210 to 280 mM NaCI, such as 220 to 270 mM NaCI, such as 230 to 240 mM NaCI.

74. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising NaCI, such as 1 to 20 mM NaCI, such as 1 to 15 mM NaCI, such as 5 to 15 mM NaCI, such as 6 to 14 mM NaCI, such as 7 to 13 mM NaCI, such as 6 to 12 mM NaCI, such as 8 to 12 mM NaCI, such as 9 to 11 mM NaCI.

75. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising KCI, such as 1 to 1000 mM KCI, such as 1 to 900 mM KCI, such as 1 to 800 mM KCI, such as 1 to 700 mM KCI, such as 1 to 600 mM KCI, such as 1 to 600 mM KCI, such as 1 to 600 mM KCI, such as 1 to 500 mM KCI, such as 1 to 400 mM KCI, such as 1 to 300 mM KCI, such as 1 to 200 mM KCI, such as 10 to 200 mM KCI, such as 20 to 200 mM KCI, such as 30 to 200 mM KCI, such as 40 to 200 mM KCI, such as 50 to 200 mM KCI, such as 50 to 150 mM KCI.

76. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising KCI, such as 1 to 20 mM KCI, such as 1 to 15 mM KCI, such as 5 to 15 mM KCI, such as 6 to 14 mM KCI, such as 7 to 13 mM KCI, such as 6 to 12 mM KCI, such as 8 to 12 mM KCI, such as 9 to 11 mM KCI77. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising KCI, such as 0.1 to 10 mM KCI, 0.1 to 5 mM KCI, 0.1 to 2 mM KCI, 0.1 to 1 mM KCI, 0.1 to 0.5 mM KCI.

78. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MgCh, such as 0.1 to 20 mM MgCh, such as 0.5 to 20 mM MgCh, such as 1 to 20 mM MgCh, such as 1 to 10 mM MgCh, such as 1 to 9 mM MgCh, such as 1 to 8 mM MgCh, such as 1 to 7 mM MgCh, such as 1 to 6 mM MgCh, such as 1 to 5 mM MgCh, such as 1 to 4 mM MgCh, such as 1 to 3 mM MgCh.

79. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising CaCh, such as 0.1 to 20 mM CaCh, such as 0.5 to 20 mM CaCh, such as 1 to20 mM CaCh, such as 1 to 10 mM CaCh, such as 1 to 9 mM CaCh, such as 1 to 8 mM CaCh, such as 1 to 7 mM CaCh, such as 1 to 6 mM CaCh, such as 1 to 5 mM CaCh, such as 1 to 4 mM CaCh, such as 1 to 3 mM CaCh.

80. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI, such as 1 to 100 mM Tris-CI, such as 1 to 90 mM Tris-CI, such as 1 to 80 mM Tris-CI, such as 1 to 70 mM Tris-CI, such as 10 to 70 mM Tris-CI, such as 20 to 70 mM Tris-CI, such as 30 to 70 mM Tris-CI, such as 40 to 60 mM Tris-CI.81 . The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI, such as 1 to 50 mM Tris-CI, such as 1 to 40 mM Tris-CI, such as 1 to 30 mM Tris-CI, such as 5 to 30 mM Tris-CI, such as 10 to 30 mM Tris-CI, such as 15 to 25 mM Tris-CI.

82. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Guanidine HCI, such as 0.1 to 5 mM Guanidine HCI, such as 0.1 to 4 mM Guanidine HCI, such as 0.1 to 3 mM Guanidine HCI, such as 0.1 to 2 mM Guanidine HCI, such as 0.5 to 1 .5 mM Guanidine HCI.

83. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, such as 0.01 to 5 % non-ionic surfactant, such as 0.01 to 4 % non-ionic surfactant, such as 0.01 to 3 % non-ionic surfactant, such as 0.01 to 2 % non-ionic surfactant, such as 0.1 to 2 % non- ionic surfactant, such as 0.5 to 2 % non-ionic surfactant, such as 0.5 to 1 .5 % non-ionic surfactant, such as 0.5 to 1 % non-ionic surfactant.

84. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising DTT, such as 0.01 to 1 mM DTT, such as 0.01 to 0.5 mM DTT, such as 0.01 to 0.4 mM DTT, such as 0.01 to 0.3 mM DTT, such as 0.01 to 0.2 mM DTT, such as 0.5 to 0.2 mM DTT.

85. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising arginine, such as 0.01 to 10 M arginine, such as 0.01 to 5 M arginine, such as 0.01 to 4 M arginine, such as 0.01 to 3 M arginine, such as 0.01 to 2 M arginine, such as 0.1 to 2 M arginine, such as 0.1 to 1 M arginine, such as 0.2 to 0.9 M arginine, such as 0.3 to 0.8 M arginine, such as 0.4 to 0.7 M arginine, such as 0.4 to 0.6 M arginine.

86. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising polyethylene glycol 3,550, such as 0.001 to 10 % polyethylene glycol 3,550, such as 0.001 to 5 % polyethylene glycol 3,550, such as 0.001 to 1 % polyethylene glycol 3,550, such as 0.001 to 0.5 % polyethylene glycol 3,550, such as 0.01 to 0.5 % polyethylene glycol 3,550, such as 0.01 to 0.1 % polyethylene glycol 3,550.

87. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising GSH, such as 0.01 to 10 mM GSH, such as 0.01 to 5 mM GSH, such as 0.01 to 1 mM GSH, such as 0.01 to 0.5 mM GSH, such as 0.05 to 0.5 mM GSH, such as 0.05 to 0.4 mM GSH, such as 0.05 to 0.3 mM GSH, such as 0.05 to 0.2 mM GSH.

88. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising GSSH, such as 0.001 to 1 mM GSSH, such as 0.001 to 0.5 mM GSSH, such as 0.001 to 0.4 mM GSSH, such as 0.001 to 0.3 mM GSSH, such as 0.001 to 0.2 mM GSSH, such as 0.001 to 0.1 mM GSSH, such as 0.005 to 0.1 mM GSSH, such as 0.005 to 0.05 mM GSSH.

89. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising EDTA, such as 0.1 to 10 mM EDTA, such as 0.1 to 5 mM EDTA, such as 0.1 to 4 mM EDTA, such as 0.5 to 4 mM EDTA, such as 0.5 to 3 mM EDTA, such as 0.5 to 2 mM EDTA.

90. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising sucrose, such as 0.01 to 10 M sucrose, such as 0.01 to 5 M sucrose, such as 0.01 to 2 M sucrose, such as 0.01 to 1 M sucrose, such as 0.1 to 1 M sucrose, such as 0.01 to 0.9 M sucrose, such as 0.01 to 0.8 M sucrose, such as 0.01 to 0.8 M sucrose, such as 0.1 to 0.7 M sucrose, such as 0.1 to 0.6 M sucrose, such as 0.2 to 0.6 M sucrose, such as 0.3 to 0.6 M sucrose, such as 0.3 to 0.5 M sucrose.91 . The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising urea, such as 1 to 15 M urea, 2 to 14 M urea, 3 to 13 M urea, 4 to 12 M urea, 5 to 11 M urea, 6 to 10 M urea or 7 to 9 M urea.

92. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Na2HPO4, such as 1 to 100 mM Na2HPO4, such as 1 to 90 mM Na2HPO4, such as 1 to 80 mM Na2HPO4, such as 1 to 70 mM Na2HPO4, such as 1 to 60 mM Na2HPO4, such as 1 to 50 mM Na2HPO4, such as 1 to 40 mM Na2HPO4, such as 1 to 30 mM Na2HPO4, such as 1 to 20 mM Na2HPO4, such such as 5 to 15 mM Na2HPO4.

93. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising KH2PO4, such as 1 to 100 mM KH2PO4, such as 1 to 90 mM KH2PO4, such as 1 to 80 mM KH2PO4, such as 1 to 70 mM KH2PO4, such as 1 to 60 mM KH2PO4, such as 1 to 50 mM KH2PO4, such as 1 to 40 mM KH2PO4, such as 1 to 30 mM KH2PO4, such as 1 to 20 mM KH2PO4, such as 5 to 20 mM KH2PO4, such as 5 to 15 mM KH2PO4.

94. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, MgCh and / or CaCh, such as 1 to 100 mM MES pH 6.0, 1 to 20 mM NaCI, 0.01 to 5 mM KCI, 0.1 to 10 mM MgCh and / or 0.1 to 10 mM CaCh, such as 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCI, 0.1 to 1 mM KCI, 0.5 to 4 mM MgCh and / or 0.5 to 4 mM CaCh.

95. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI and / or KCI, such as 1 to 100 mM MES pH 6.0, 200 to 300 mM NaCI and / or 1 to 50 mM KCI, such as 30 to 70 mM MES pH 6.0, 220 to 260 mM NaCI and / or 1 to 20 mM KCI,96. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, MgCh, CaCh, Guanidine HCI, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or DTT.

97. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, MgCh, CaCh, arginine, polyethylene glycol 3,550, GSH and / or GSSH.

98. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, EDTA, KCI, sucrose, M Guanidine HCI, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550 and / or DTT.

99. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, MgCh, CaCh, arginine, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, GSH and / or mM GSSH.

100. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, EDTA, sucrose, Guanidine HCI and / or DTT.

101. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, EDTA, arginine, sucrose, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550, GSH and / or GSSH.

102. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising MES pH 6.0, NaCI, KCI, MgCh, CaCh, Guanidine HCI, polyethylene glycol 3,550 and / or DTT.

103. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising mM Tris-CI pH 8.5, NaCI, KCI, MgCh, CaCh, sucrose, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550, GSH and GSSH.

104. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, NaCI, KCI, EDTA, arginine, Guanidine HCI, polyethylene glycol 3,550 and / or DTT.

105. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, NaCI, KCI, MgCh, CaCh, arginine, sucrose, Guanidine HCI, mM GSH and / or GSSH.

106. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, mM NaCI, KCI, EDTA, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or DTT.

107. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, NaCI, KCI, EDTA, polyethylene glycol 3,550, GSH and / or GSSH.

108. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, NaCI, KCI, 1 mM EDTA, arginine, Guanidine HCI, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or DTT.

109. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, NaCI, KCI, MgCh, CaCh, arginine, sucrose, Guanidine HCI, non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, polyethylene glycol 3,550, GSH and / or GSSH.

110. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI pH 8.5, NaCI, KCI, MgCh, CaCh, sucrose and / or DTT.

111. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising Tris-CI and / or urea, having pH 8.0.

112. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising NaCI, KCI, Na2HPO4 and / or 10 mM KH2PO4, having pH 8.0.

113. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCI, 0.1 to 1 mM KCI, 1 to 5 mM MgCh, 1 to 5 mM CaCh, 0.5 to 1 M Guanidine HCI, 0.1 to 2 % non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or 0.01 to 0.5 mM DTT.

114. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCI, 0.1 to 1 mM KCI, 1 to 5 mM MgCh, 1 to 5 mM CaCh, 0.05 to 1 M arginine, 0.05 to 0.5 % polyethylene glycol 3,550, 0.01 to 1 mM GSH and / or 0.001 to 0.05 mM GSSH.

115. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 5 to 15 mM NaCI, 0.5 to 5 mM EDTA, 0.1 to 1 mM KCI, 0.1 to 1 M sucrose, 0.5 to 1 M Guanidine HCI, 0.05 to 1 % non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.005 to 0.1% polyethylene glycol 3,550 and / or 0.01 to 1 mM DTT.

116. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 200 to 300 mM NaCI, 1 to 20 mM KCI,1 to 5 mM MgCh, 1 to 2 mM CaCh, 0.05 to 1 M arginine, 0.05 to 1 % non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.01 to 1 mM GSH and / or 0.001 to 0.1 mM GSSH.

117. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 200 to 300 mM NaCI, 1 to 20 mM KCI, 0.5 to 5 mM EDTA, 0.1 to 1 M sucrose, 0.5 to 1 M Guanidine HCI and / or 0.01 to 1 mM DTT.

118. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70 mM MES pH 6.0, 240 mM NaCI, 10 mM KCI, 1 mM EDTA, 0.5 M arginine, 0.4 M sucrose, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.

119. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 30 to 70MES pH 6.0, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.75 M Guanidine HCI, 0.05% polyethylene glycol 3,550 and / or 0.1 mM DTT.

120. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.4 M sucrose, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.121 . The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 1 mM EDTA, 0.5 M arginine, 0.75 M Guanidine HCI, 0.05% polyethylene glycol 3,550 and / or 0.1 mM DTT.

122. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.5 M arginine, 0.4 M sucrose, 0.75 M Guanidine HCI, 0.1 mM GSH and / or 0.01 mM GSSH.

123. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 9.6 mM NaCI, 0.4 mM KCI, 1 mM EDTA, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.1 mM DTT124. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 1 mM EDTA, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.

125. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 1 mM EDTA,0.5 M arginine, 0.75 M Guanidine HCI, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, and / or 0.1 mM DTT.

126. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.5 M arginine, 0.4 M sucrose, 0.75 M Guanidine HCI, 0.5% non-ionic surfactant, such as Tergitol NP-7, Tergitol NP-10 or Triton X-100, 0.05% polyethylene glycol 3,550, 0.1 mM GSH and / or 0.01 mM GSSH.

127. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 50 mM Tris-CI pH 8.5, 240 mM NaCI, 10 mM KCI, 2 mM MgCh, 2 mM CaCh, 0.4 M sucrose and / or 0.1 mM DTT.

128. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 20 mM Tris-CI and / or 8 M urea, having pH 8.0.

129. The method according to any one of the preceding claims, wherein the assembly activator is a buffer comprising 500 mM NaCI, 100 mM KCI, 10 mM Na2HPO4 and / or 10 mM KH2PO4, having pH 8.0.

130. The method according to any one of the preceding claims, wherein the activating time is between 30 minutes and 24 hours, such between 1 hour and 24 hours, such between 1 hour and 20 hours, such between 1 hour and 16 hours.

131. The method according to any one of the preceding claims, wherein the activating time is between 15 minutes and 5 hours, such as between 30 minutes and 5 hours, such as between 30 minutes and 4 hours, such as between 30 minutes and 3 hours, such as between 30 minutes and 2 hours, such as between 30 minutes and 1 hour and 30 minutes.

132. The method according to any one of the preceding claims, wherein the activating temperature is between 1 °C and 45 °C, such as between 10 °C and 45 °C, such as between 10 °C and 40 °C, such as between 10 °C and 35 °C, such as between 10 °C and 30 °C, such as between 10 °C and 25 °C.

133. The method according to any one of the preceding claims, wherein the activating temperature is between 1 °C and 30 °C, such as between 10 °C and 30 °C, such as between 10 °C and 25 °C, such as between 15 °C and 25 °C, such as between 20 °C and 25 °C.

134. The method according to any one of the preceding claims, wherein the activating temperature is between 15 °C and 25 °C, such as between 16 °C and 25 °C, such as between 17 °C and 25 °C, such as between 17 °C and 24 °C, such as between 18 °C and 24 °C, such as between 19 °C and 24 °C, such as between 20 °C and 24 °C, such as between 21 °C and 24 °C, such as between 21 °C and 23 °C,135. The method according to any one of the preceding claims, wherein the activating temperature is between 1 °C and 10 °C, such as between 1 °C and 9 °C, such as between 1 °C and 8 °C, such as between 1 °C and 7 °C, such as between 1 °C and 6 °C, such as between 2 °C and 6 °C, such as between 3 °C and 5 °C.

136. The method according to any one of the preceding claims, wherein the viral capsid proteins further comprise a purification tag, such as a His-tag and / or a C-tag, (EPEA).

137. The method according to any one of the preceding claims, wherein activating viral capsid protein concentration is higher than the inhibitory viral capsid protein concentration.

138. The method according to any one of the preceding claims, wherein the method comprises an assembly enhancer which enhances in vitro assembly.

139. The method according to any one of the preceding claims, wherein the assembly enhancer is selected from the group consisting of membrane-associated accessory protein, assembly activation protein, an E2A transcriptional regulator, an ICP8 single-stranded DNA- binding protein, presence of any cleavable protein or amino acid sequence, an enzyme that mediates glycosylation, and / or an enzyme that mediates a post translational modification.

140. The method according to any one of the preceding claims, wherein the assembly enhancer is selected from the group consisting of membrane-associated accessory protein and / or assembly activation protein.

141. The method according to any one of the preceding claims, wherein the one or more populations of inhibited parvovirus viral capsid proteins are provided at a defined stoichiometry.

142. The method according to any one of the preceding claims, wherein the one or more populations of inhibited parvovirus viral capsid proteins comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60.

143. A population of assembled parvovirus capsids obtainable by the method according to any one of preceding claims.

144. The population of assembled parvovirus capsids according to any one of preceding claims, wherein the population of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60.

145. The population of assembled parvovirus capsids according to any one of preceding claims, wherein the population of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in a ratio of 1 :1 :10 (VP1 :VP2:VP3).

146. The method according to any one of the preceding claims, wherein the nucleic acid molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.

147. The method according to any one of the preceding claims, wherein the nucleic acid molecule is a viral nucleic acid molecule and / or a non-viral nucleic acid molecule.

148. The method according to any one of the preceding claims, wherein the nucleic acid molecule is a viral nucleic acid molecule.

149. The method according to any one of the preceding claims, wherein the nucleic acid molecule is a non-viral nucleic acid molecule.

150. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprises deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA).

151. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprises DNA.

152. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprises RNA.

153. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprises AAV DNA.

154. The method according to any one of the preceding claims, wherein the reaction mixture comprises adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), a pH buffer, such as HEPES or MOPS buffer, dithiothreitol, creatine phosphate, creatine phosphokinase (CPK), and / or magnesium chloride.

155. The method according to any one of the preceding claims, wherein the reaction mixture comprises adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), a pH buffer, such as HEPES or MOPS buffer, dithiothreitol, and / or magnesium chloride.

156. The method according to any one of the preceding claims, wherein the filling conditions are selected from the group consisting of ATP concentration, filling temperature, filling pH, CPK concentration, an ATP regeneration system, parvovirus capsid concentration and / or filling time.

157. The method according to any one of the preceding claims, wherein the filling conditions are selected from the group consisting of ATP concentration, filling temperature, filling pH, CPK concentration an ATP regeneration system, and / or parvovirus capsid concentration.

158. The method according to any one of the preceding claims, wherein the filling conditions comprise ATP concentration.

159. The method according to any one of the preceding claims, wherein the ATP concentration is between 0.1 and 181.44 mM, such as between 0.1 and 180 mM, such as between 0.1 and 170 mM, such as between 0.1 and 160 mM, such as between 0.1 and 150 mM, such as between 0.1 and 140 mM, such as between 0.1 and 130 mM, such as between 0.1 and 120 mM, such as between 0.1 and 110 mM, such as between 0.1 and 100 mM.

160. The method according to any one of the preceding claims, wherein the ATP concentration is between 0.1 and 100 mM. such as between 1 and 90 mM, such as between 1 and 80 mM, such as between 1 and 70 mM, such as between 1 and 60 mM, such as between 1 and 50 mM, such as between 1 and 40 mM, such as between 1 and 30 mM, such as between 1 and 20 mM, such as between 1 and 15 mM, such as between 1 and 10 mM.

161. The method according to any one of the preceding claims, wherein the ATP concentration is between 0.01 and 10 mM, such as between 0.05 and 10 mM, such as between 0.05 and 9 mM, such as between 0.05 and 8 mM, such as between 0.05 and 7 mM, such as between 0.05 and 6 mM, such as between 0.05 and 5 mM, such as between 0.05 and 4 mM, such as between 0.05 and 3 mM, such as between 0.05 and 2.5 mM, such as between 0.1 and 2 mM.

162. The method according to any one of the preceding claims, wherein the ATP concentration is between 0.05 and 15 mM.

163. The method according to any one of the preceding claims, wherein the filling conditions comprise filling temperature.

164. The method according to any one of the preceding claims, wherein the filling temperature is 0 °C and 89.7 °C, such as between 5 °C and 80 °C, such as between 5 °C and 70 °C, such as between 5 °C and 60 °C, between 5 °C and 50 °C, such as between 5 °C and 50 °C, such as between 10 °C and 50 °C, such as between 15 °C and 50 °C, such as between 20 °C and 50 °C, such as between 25 °C and 50 °C, such as between 30 °C and 50 °C.

165. The method according to any one of the preceding claims, wherein the filling temperature is between 1 °C and 40 °C, such as between 5 °C and 40 °C, such as between 10 °C and 40 °C, such as between 15 °C and 40 °C, such as between 20 °C and 40 °C, such as between 25 °C and 40 °C, such as between 26 °C and 40 °C, such as between 27 °C and 40 °C, such as between 28 °C and 40 °C, such as between 29 °C and 40 °C, such as between 29 °C and 39 °C.

166. The method according to any one of the preceding claims, wherein the filling temperature is between 20 °C and 50 °C, such as between 20 °C and 45 °C, such as between 20 °C and 40 °C, such as between 25 °C and 40 °C, such as between 26 °C and 40 °C, such as between 27 °C and 40 °C, such as between 28 °C and 40 °C, such as between 29 °C and 40 °C, such as between 29 °C and 39 °C.

167. The method according to any one of the preceding claims, wherein the filling temperature is between 30 °C and 50 °C, such as between 30 °C and 45 °C, such as between 30 °C and 45 °C, such as between 31 °C and 45 °C, such as between 32 °C and 45 °C, such as between 33 °C and 45 °C, such as between 34 °C and 45 °C, such as between 30 °C and 44 °C, such as between 30 °C and 43 °C, such as between 30 °C and 42 °C, such as between 30 °C and 41 °C.

168. The method according to any one of the preceding claims, wherein the filling temperature is between 33 °C and 42 °C, such as between 34 °C and 41 °C.

169. The method according to any one of the preceding claims, wherein the filling temperature is between 35 °C and 40 °C.

170. The method according to any one of the preceding claims, wherein the filling conditions comprise filling pH.

171. The method according to any one of the preceding claims, wherein the filling pH is between 3 and 10, such as between 4 and 9, such as between 4.5 and 9.

172. The method according to any one of the preceding claims, wherein the filling pH is between 5 and 8.8, such as between 5.5 and 8.8, such as between 6 and 8.8, such as between 6 and 8.5, such as between 6 and 8.

173. The method according to any one of the preceding claims, wherein the filling pH is between 6 and 7.5.

174. The method according to any one of the preceding claims, wherein the filling conditions comprise ATP regenerating system, such as acetate kinase using acetyl phosphate as phosphate donor, creatine kinase using creatine phosphate as phosphate donor, pyruvate kinase using phosphoenolpyruvate as phosphate donor, and polyphosphate kinase using polyphosphate as phosphate donor.

175. The method according to any one of the preceding claims, wherein the filling conditions comprise the CPK concentration.

176. The method according to any one of the preceding claims, wherein the CPK concentration is between 1 and 1000 ug / ml, such as between 1 and 500 ug / ml, such as between 1 and 400 ug / ml, such as between 1 and 300 ug / ml, such as between 1 and 200 ug / ml, such as between 1 and 100 ug / ml.

177. The method according to any one of the preceding claims, wherein the CPK concentration is between 1 and 100 ug / ml, such as between 1 and 90 ug / ml, such as between 1 and 80 ug / ml, such as between 1 and 70 ug / ml, such as between 1 and 60 ug / ml, such asbetween 1 and 50 ug / ml, such as between 1 and 40 ug / ml, such as between 1 and 30 ug / ml, such as between 1 and 20 ug / ml, such as between 1 and 10 ug / ml.

178. The method according to any one of the preceding claims, wherein the CPK concentration is between 5 and 110 ug / ml, such as between 5 and 100 ug / ml, between 5 and 90 ug / ml.

179. The method according to any one of the preceding claims, wherein the CPK concentration is between 10 and 100 ug / ml.

180. The method according to any one of the preceding claims, wherein the filling conditions comprise the parvovirus capsid concentration.

181. The method according to any one of the preceding claims, wherein the parvovirus capsid concentration is between 1 e4 and 5 e16 / mL, such as between 1 e4 and 5 e15 / mL, such as between 1e4 and 1 e15 / mL, such as between 1 e4 and 1 e15 / mL, such as between 1e4 and 1 e14 / mL, such as between 1e4 and 1 e13 / mL, such as between 1e4 and 1 e12 / mL, such as between 1e4 and 1 e11 / mL, such as between 1 e4 and 1 e10 / mL, such as between 1e4 and 1 e9 / mL, such as between 1e4 and 1 e8 / mL, such as between 1e4 and 1 e7 / mL, such as between 1e4 and 1 e6 / mL, such as between 1 e4 and 1 e5 / mL.

182. The method according to any one of the preceding claims, wherein the parvovirus capsid concentration is between 0.01 and 100 e10 / mL, such as between 0.1 and 100 e10 / mL, such as between 0.1 and 90 e10 / mL. such as between 0.1 and 80 e10 / mL. such as between 0.1 and 70 e10 / mL. such as between 0.1 and 60 e10 / mL. such as between 0.1 and 50 e10 / mL. such as between 0.1 and 40 e10 / mL. such as between 0.1 and 30 e10 / mL. such as between 0.1 and 20 e10 / mL. such as between 0.1 and 10 e10 / mL. such as between 0.1 and 5 e10 / mL.

183. The method according to any one of the preceding claims, wherein the parvovirus capsid concentration is between 0.01 and 10 e10 / mL, such as between 0.1 and 10 e10 / mL, such as between 0.5 and 10 e10 / mL, such as between 0.5 and 9 e10 / mL, such as between 0.5 and 8 e10 / mL, such as between 0.5 and 7 e10 / mL, such as between 0.5 and 6 e10 / mL, such as between 0.5 and 5 e10 / mL.

184. The method according to any one of the preceding claims, wherein the parvovirus capsid concentration is between 0.5 and 7 e10 / mL.

185. The method according to any one of the preceding claims, wherein the reaction mixture comprises HEPES, such as 30mM HEPES, MES buffer, phosphate buffer, Tris buffer or MOPS buffer.

186. The method according to any one of the preceding claims, wherein the filling conditions comprises HEPES, such as 30mM HEPES, MES buffer, phosphate buffer, Tris buffer or MOPS buffer.

187. The method according to any one of the preceding claims, wherein the filling conditions comprises the following: a. the ATP concentration is between 0.05 and 15 mM; b. the filling temperature is between 35 °C and 40 °C; c. the CPK concentration is between 10 and 100 ug / ml; and / or d. the parvovirus capsid concentration is between 0.5 and 7 e10 / mL.

188. The method according to any one of the preceding claims, wherein the filling conditions comprises filling time.

189. The method according to any one of the preceding claims, wherein the filling time is between 0.5 and 24 hours, such as between 0.5 and 20 hours, such as between 0.5 and 16 hours, such as between 0.5 and 12 hours, such as between 0.5 and 10 hours, such as between 1 and 10 hours, such as between 1 and 9 hours, such as between 2 and 8 hours, such as between 2 and 7 hours, such as between 3 and 6 hours, such as between 3 and 5 hours.

190. The method according to any one of the preceding claims, wherein the filling time is between 1 and 7 hours, such as between 2 and 7 hours, such as between 2.5 and 6.5 hours, such as between 3 and 6 hours, such as between 3 and 5 hours, such as between 3.5 and 4.5 hours.

191. The method according to any one of the preceding claims, wherein the filling time is between 3 and 5 hours.

192. The method according to any one of the preceding claims, wherein the filling conditions comprises the following: a. the ATP concentration is between 0.05 and 15 mM; b. the filling temperature is between 35 °C and 40 °C; c. the CPK concentration is between 10 and 100 ug / ml;d. the parvovirus capsid concentration is between 0.5 and 7 e10 / mL; and / or e. the filling time is between 3 and 5 hours.

193. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprises a first inverted terminal repeat (ITR1) and / or a second inverted terminal repeat (ITR2).

194. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 is from AAV.

195. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 comprises the Rep binding domain.

196. The method according to any one of the preceding claims, wherein the nucleic acid molecule, the ITR1 and / or the ITR2 are operably linked to each other in a 5’-to-3’ direction.

197. The method according to any one of the preceding claims, wherein the nucleic acid molecule, the ITR1 and / or the ITR2 are operably linked to each other in a 3’-to-5’ direction198. The method according to any one of the preceding claims, wherein the method comprises a filling enhancer which enhances in vitro filling.

199. The method according to any one of the preceding claims, wherein the filling enhancer is selected from the group consisting of membrane-associated accessory protein, assembly activation protein, an E2A transcriptional regulator, an ICP8 single-stranded DNA-binding protein, presence of any cleavable protein or amino acid sequence, an enzyme that mediates glycosylation, and / or an enzyme that mediates a post translational modification.

200. The method according to any one of the preceding claims, wherein the filling enhancer is selected from the group consisting of membrane-associated accessory protein and / or assembly activation protein.201 . The method according to any one of the preceding claims, wherein the E2A transcriptional regulator is from adenovirus.

202. The method according to any one of the preceding claims, wherein the ICP8 singlestranded DNA-binding protein is from herpes virus.

203. The method according to any one of the preceding claims, wherein the enzyme that mediates a post translational modification is synthetic.

204. The method according to any one of the preceding claims, wherein the filling conditions are conditions which denature the assembled capsids.

205. The method according to any one of the preceding claims, wherein the filling conditions which denature are condition factors selected from this list comprising: pH, sonication, ionic strength / conductivity, or temperature.

206. The method according to any one of the preceding claims, wherein the filled parvovirus capsids comprises an increased amount of intact nucleic acid molecule compared to parvovirus capsids that have been filled using conventional methods207. The method according to any one of the preceding claims, wherein the filled parvovirus capsids comprise an 80% intact nucleic acid molecule, such as an 85% intact nucleic acid molecule, such as a 90% intact nucleic acid molecule, such as a 95% intact nucleic acid molecule, such as a 96% intact nucleic acid molecule, such as a 97% intact nucleic acid molecule, such as a 98% intact nucleic acid molecule, such as a 99% intact nucleic acid molecule, such as a 100% intact nucleic acid molecule.

208. The method according to any one of the preceding claims, wherein the filled parvovirus capsids comprise a 100% intact nucleic acid molecule.

209. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprised in the filled parvovirus capsids has reduced truncation compared to a nucleic acid molecule comprised in parvovirus capsid that have been filled using conventional methods.

210. The method according to any one of the preceding claims, wherein the nucleic acid molecule comprised in the filled parvovirus capsids has 20% truncation, such as 15% truncation, such as 10% truncation, such as 5% truncation, such as 4% truncation, such as 3% truncation, such as 2% truncation, such as 1 % truncation, such as 0% truncation.211 . The method according to any one of the preceding claims, wherein the nucleic acid molecule comprised in the filled parvovirus capsids has no truncation.

212. The method according to any one of the preceding claims, wherein the nucleic acid comprises the ITR1 , the transgene of interest and the ITR2.

213. A population of filled parvovirus capsids obtainable by the method according to any one of preceding claims.

214. The population of filled parvovirus capsids according to any one of preceding claims, wherein the population of filled parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60.

215. The population of filled parvovirus capsids according to any one of preceding claims, wherein the population of filled parvovirus capsids comprises VP1 , VP2 and VP3 in a ratio of 1 :1 :10 (VP1 :VP2:VP3).

216. The method according to any one of the preceding claims, wherein the one or more populations of inhibited parvovirus viral capsid proteins and / or the one or more populations of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in any ratio of integers where VP1 + VP2 + VP3 = 60.

217. The method according to any one of the preceding claims, wherein the one or more populations of inhibited parvovirus viral capsid proteins and / or the one or more populations of assembled parvovirus capsids comprises VP1 , VP2 and VP3 in a ratio of 1 :1 :10 (VP1 :VP2:VP3).

218. The method according to any one of the preceding claims, wherein the method further comprises a step of purifying the assembled parvovirus capsids and / or the filled parvovirus capsids.

219. The method according to any one of the preceding claims, wherein the step of purifying comprises chromatography, ultra-centrifugation, centrifugation, flocculation, filtration, and / or ultrafiltration / diafiltration.

220. The method according to any one of the preceding claims, wherein the method further comprises a step of formulating the assembled parvovirus capsids and / or the filled parvovirus capsids into a formulation, optionally wherein the assembled parvovirus capsids and / or the filled parvovirus capsids are purified assembled parvovirus capsids and / or purified filled parvovirus capsids.221 . The method according to any one of the preceding claims, wherein the step of formulating comprises formulating the assembled parvovirus capsids and / or the filled parvovirus capsids into a formulation with trehalose, sugar, sucrose, polyols, and / or dimethylsulfoxide.

222. The method according to any one of the preceding claims, wherein the step of formulating comprises formulating the assembled parvovirus capsids and / or the filled parvovirus capsids into a formulation with a pharmaceutically acceptable carrier, diluent and / or excipient.

223. The method according to any one of the preceding claims, wherein the pharmaceutically acceptable carrier, diluent and / or excipient is selected from the group consisting of tween 20, tween 80, trehalose, Pluronic F68, sorbitol and / or glycerol, such as 0.0001 to 0.01 % Pluronic F68, 1 to 10 % sorbitol and / or 1 to 10% glycerol.

224. The method according to any one of the preceding claims, wherein the method further comprises a step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids, optionally wherein the storing is under conditions that maintain capsid integrity.

225. The method according to any one of the preceding claims, wherein the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids is at room temperature.

226. The method according to any one of the preceding claims, wherein the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids is at 1 °C to 25 °C, such as 1 °C to 20 °C, such as 5 °C to 25 °C, such as 10 °C to 25 °C, such as 15 °C to 25 °C.

227. The method according to any one of the preceding claims, wherein the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids comprises lyophilizing and aerosolizing the assembled parvovirus capsids and / or the filled parvovirus capsids.

228. The method according to any one of the preceding claims, wherein the step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids at a low pH or a high pH.

229. The method according to any one of the preceding claims, comprising step of storing the assembled parvovirus capsids and / or the filled parvovirus capsids under pressure.

230. The method according to any one of the preceding claims, wherein the VP1 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10.231 . The method according to any one of the preceding claims, wherein the VP1 comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10.

232. The method according to any one of the preceding claims, wherein the VP1 comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10, optionally wherein the VP1 comprises an amino acid sequence that is at least 96%, 97%, 98%, and 99% identical to the amino acid sequences of SEQ ID NOs: 1-4 or 7-10.

233. The method according to any one of the preceding claims, wherein the VP1 comprises an amino acid sequence that is identical to any one of the amino acid sequences of SEQ ID NOs: 1- 4 or 7-10.

234. The method according to any one of the preceding claims, wherein the VP1 has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10.

235. The method according to any one of the preceding claims, wherein the VP1 has an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7-10.

236. The method according to any one of the preceding claims, wherein the VP1 has an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 2 or 4, optionally wherein the VP1 has an amino acid sequence that is at least 96%, 97%, 98%, and 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1-4 or 7- 10.

237. The method according to any one of the preceding claims, wherein the VP1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 or 7-10.

238. The method according to any one of the preceding claims, wherein the VP2 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

239. The method according to any one of the preceding claims, wherein the VP2 comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

240. The method according to any one of the preceding claims, wherein the VP2 comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17, optionally wherein the VP2 comprises an amino acid sequence that is at least 96%, 97%, 98%, and 99% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11 -17.241 . The method according to any one of the preceding claims, wherein the VP2 comprises an amino acid sequence that is identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

242. The method according to any one of the preceding claims, wherein the VP2 has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

243. The method according to any one of the preceding claims, wherein the VP2 has an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11-17.

244. The method according to any one of the preceding claims, wherein the VP2 has an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 13, 16, or 17, optionally wherein the VP2 has an amino acid sequence that is at least 96%, 97%, 98%, and 99% identical to any one of the amino acid sequences of SEQ ID NOs: 5 or 11 -17.

245. The method according to any one of the preceding claims, wherein the VP2 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 or 11-17.

246. The method according to any one of the preceding claims, wherein the VP3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

247. The method according to any one of the preceding claims, wherein the VP3 comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

248. The method according to any one of the preceding claims, wherein the VP3 comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24, optionally wherein the VP3 comprises an amino acid sequence that is at least 96%, 97%, 98%, and 99% identical to any one of the amino acid sequences of SEQ ID NOs: 6 and 18-24.

249. The method according to any one of the preceding claims, wherein the VP3 comprises an amino acid sequence that is identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

250. The method according to any one of the preceding claims, wherein the VP3 has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

251. The method according to any one of the preceding claims, wherein the VP3 has an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

252. The method according to any one of the preceding claims, wherein the VP3 has an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24, optionally wherein the VP3 has an amino acid sequence that is at least 96%, 97%, 98%, and 99% identical to any one of the amino acid sequences of SEQ ID NOs: 6 or 18-24.

253. The method according to any one of the preceding claims, wherein the VP3 has an amino acid sequence of SEQ ID NO: 6 SEQ ID NOs: 6 or 18-24.

254. The method according to any one of the preceding claims, wherein Rep protein is a synthetic Rep protein.

255. The method according to any one of the preceding claims, wherein Rep protein is a recombinant Rep protein and / or a recombinantly produced Rep protein.

256. The method according to any one of the preceding claims, the Rep protein comprises multiple Rep proteins, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 Rep proteins.

257. The method according to any one of the preceding claims, the step of providing a Rep protein is a step of providing multiple Rep proteins, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 Rep proteins258. The method according to any one of the preceding claims, wherein the Rep protein is from a Parvoviridae.

259. The method according to any one of the preceding claims, wherein the Rep protein is from a Densovirinae, Hamaparvovirinae, or Parvovirinae.

260. The method according to any one of the preceding claims, wherein the Rep protein is from is a Amdoparvovirus, Artiparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetraparvovirus.261 . The method according to any one of the preceding claims, wherein the Rep protein is from a Dependoparvovirus.

262. The method according to any one of the preceding claims, wherein the Rep protein is from an AAV.

263. The method according to any one of the preceding claims, wherein the Rep protein is synthetic Rep protein and / or a recombinant Rep protein.

264. The method according to any one of the preceding claims, wherein the Rep protein is selected from the group consisting of AAV Rep40, AAV Rep52, AAV Rep68, AAV Rep78.

265. The method according to any one of the preceding claims, wherein the Rep40 is from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

266. The method according to any one of the preceding claims, wherein the Rep52 is from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6,serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

267. The method according to any one of the preceding claims, wherein the Rep68 is from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

268. The method according to any one of the preceding claims, wherein the Rep78 is from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

269. The method according to any one of the preceding claims, wherein the Rep protein is selected from the list consisting of: a. AAV Rep40; b. AAV Rep52; c. AAV Rep40 and AAV Rep52; d. AAV Rep68; e. AAV Rep78; f. AAV Rep68 and AAV Rep78; g. AAV Rep40, AAV Rep52, AAV Rep68, and AAV Rep78; h. AAV Rep40, AAV Rep52, and AAV Rep68; and / or i. AAV Rep40, AAV Rep52, and AAV Rep78.

270. The method according to any one of the preceding claims, wherein the Rep40, Rep52, Rep68, and / or Rep78 are from different AAV serotypes.

271. The method according to any one of the preceding claims, wherein the Rep40, Rep52, Rep68, and / or Rep78 are from the same AAV serotype.

272. The method according to any one of the preceding claims, wherein the Rep protein is produced in an eukaryotic microbial system, such as yeast, such as a methylotrophic yeast.

273. The method according to any one of the preceding claims, wherein the Rep protein is produced in an eukaryotic microbial system, such as P. pastoris or S. cerevisiae.

274. The method according to any one of the preceding claims, wherein the Rep protein is produced in an prokaryotic microbial system, such as E. Coli.

275. The method according to any one of the preceding claims, wherein the Rep protein is produced in a mammalian cell system, such as HEK, CHO, HeLa or Vero cells, an insect cell system, such as Sf9, Sf21 or High Five cells, a plant system, such as tobacco BY, an algae system, a metazoan cell system, a zebrafish and / or cell-free expression systems.

276. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from a Parvoviridae.

277. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from a Densovirinae, Hamaparvovirinae, or Parvovirinae.

278. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from a Amdoparvovirus, Arti parvovirus, Aveparvovirus, Bocaparvovirus, Copiparvoviru, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus, or Tetraparvovirus.

279. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from a Dependoparvovirus.

280. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from an AAV.281 . The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from AAV serotype 1 , serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 6.2, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, serotype rh10, or serotype rh74.

282. The method according to any one of the preceding claims, wherein the ITR1 and / or the ITR2 are from AAV serotype 2.

283. The method according to any one of the preceding claims, wherein the VP1 , VP2 and / or VP3, the Rep protein, and / or ITR1 and / or ITR2 are from different AAV serotypes.

284. The method according to any one of the preceding claims, wherein the VP1 , VP2 and / or VP3, the Rep protein, and / or ITR1 and / or ITR2 are from the same AAV serotype.

285. An AAV Rep fusion protein comprising: a. An AAV Rep domain or a AAV Rep protein, and b. A cleavable amino acid sequence, such as a SUMO tag.

286. The AAV Rep fusion protein according to any one of the preceding claims, wherein the AAV Rep protein is selected from the group consisting of Rep40, Rep52, Rep68 and / or Rep78.

287. The AAV Rep fusion protein according to any one of the preceding claims, further comprising a purification tag, such as a His-tag, a C-tag (EPEA), a streptavidin tag and / or biotin tag.

288. A vector encoding the AAV Rep fusion protein according to any one of the preceding claims.

289. A method of producing the AAV Rep fusion protein according to any one of the preceding claims, comprising the steps of: a. Transforming a prokaryotic microbial system, such as E. coli, with the vector of claim 288; and b. Incubating the prokaryotic microbial system, such as E. coli, thereby producing the AAV Rep fusion protein.

290. A method of producing the AAV Rep fusion protein according to any one of the preceding claims, comprising the steps of: a. Transforming an eukaryotic microbial system, such as P. pastoris or S. cerevisiae, with the vector of claim 288, b. Incubating the eukaryotic microbial system, such as yeast, such as P. pastoris, or S. cerevisiae, thereby producing the AAV Rep fusion protein.291 . The method according to claim 289 to 290, further comprising a step of inoculating the prokaryotic microbial system or the eukaryotic microbial system.

292. The method according to any one of claims 289 to 291 , further comprising a step of purifying the fusion protein.

293. The method according to any one of claims 289 to 292, wherein the eukaryotic microbial system is a methylotrophic yeast.

294. An AAV VP fusion protein comprising: a. An AAV VP domain or an AAV VP protein, and b. A cleavable amino acid sequence, such as a SUMO tag.

295. The AAV VP fusion protein according to any one of the preceding claims, wherein the AAV VP protein is selected from the group consisting of AAV VP1 , AAV VP2, and / or AAV VP.

296. The AAV VP fusion protein according to any one of the preceding claims, further comprising a purification tag, such as a His-tag, a C-tag (EPEA), a streptavidin tag and / or biotin tag.

297. The AAV VP fusion protein according to any one of the preceding claims, comprising a. A purification tag, such as a His-tag, b. A cleavable amino acid sequence, such as a SUMO tag, c. An AAV VP protein, such as AAV VP1 , AAV VP2 and / or AAV VP3, d. A thrombin cleavage site, e. A cleavable amino acid sequence, such as a SUMO tag, and f. A purification tag, such as C-tag (EPEA).

298. The AAV VP fusion protein according to any one of the preceding claims, wherein the VP protein is as defined in any one of the preceding claims.

299. A vector encoding the AAV VP fusion protein according to any one of the preceding claims.

300. A method of producing the AAV VP fusion protein according to any one of the preceding claims, comprising the steps of: a. Transforming a prokaryotic microbial system, such as E. coli, with the vector of claim 299; and b. Incubating the prokaryotic microbial system, such as E. coli, thereby producing the AAV VP fusion protein.301 . A method of producing the AAV VP fusion protein according to any one of the preceding claims, comprising the steps of:a. Transforming a eukaryotic microbial system, such as yeast, such as P. pastoris or S. cerevisiae, with the vector of claim 299, b. Incubating the eukaryotic microbial system, such as P. pastoris or S. cerevisiae, thereby producing the AAV fusion protein.

302. The method according to any one of claims 300 to 301 , further comprising a step of inoculating the eukaryotic microbial system.

303. The method according to any one of claims 300 to 302, further comprising a step of purifying the fusion protein.

304. The method according to any one of claims 300 to 303, wherein the eukaryotic microbial system is a methylotrophic yeast.

305. A method of producing the AAV Rep fusion protein or the AAV VP fusion protein according to any one of the preceding claims, comprising the steps of: a. Transforming an mammalian system, such as HEK cells, with the vector according to any one of the preceding claims, b. Incubating the mammalian system, such as HEK cells, thereby producing the AAV Rep fusion protein or the AAV VP fusion protein.

306. A kit of parts comprising: a. one or more populations of inhibited parvovirus viral capsid proteins according to any one of the preceding claims; b. an assembly inhibitor according to any one of the preceding claims; c. an assembly activator according to any one of the preceding claims; and d. optionally instructions for use.

307. A kit of parts comprising: a. one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins according to any one of the preceding claims; b. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain according to any one of the preceding claims; c. a Rep protein according to any one of the preceding claims; and d. optionally instructions for use.

308. A kit of parts comprising:a. one or more populations of assembled parvovirus capsids comprising parvovirus viral capsid proteins according to any one of the preceding claims; b. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain according to any one of the preceding claims; c. a Rep protein according to any one of the preceding claims; d. a reaction mixture according to any one of the preceding claims; and e. optionally instructions for use.

309. A kit of parts comprising: a. one or more populations of inhibited parvovirus viral capsid proteins according to any one of the preceding claims; b. an assembly inhibitor according to any one of the preceding claims; c. an assembly activator according to any one of the preceding claims; d. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain according to any one of the preceding claims; e. a Rep protein according to any one of the preceding claims; and f. optionally instructions for use.

310. A kit of parts comprising: a. one or more populations of inhibited parvovirus viral capsid proteins according to any one of the preceding claims; b. an assembly inhibitor according to any one of the preceding claims; c. an assembly activator according to any one of the preceding claims; d. a nucleic acid molecule comprising a transgene of interest and a Rep binding domain according to any one of the preceding claims; e. a Rep protein according to any one of the preceding claims; f. a reaction mixture according to any one of the preceding claims; and g. optionally instructions for use.311 . The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 35, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35.

312. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 36, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36.

313. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 37, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37.

314. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 38, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 38.

315. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 39, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 39.

316. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 40, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 40.

317. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 41 , optionally wherein the Rep protein comprises an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 41.

318. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 42, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42.

319. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 43, optionally wherein the Rep protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43.

320. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 35.321 . The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 36.

322. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 37.

323. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 38.

324. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 39.

325. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 40.

326. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 41 .

327. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 42.

328. The method according to any one of the preceding claims, wherein the Rep protein comprises or consists of an amino acid sequence of SEQ ID NO: 43.

329. The method according to any one of the preceding claims, wherein the Rep protein concentration is between 0.01 and 100 pM, such as between 0.01 and 10 pM, between 0.01 and 5 pM, between 0.01 and 2.5 pM, between 0.01 and 1 pM, between 0.01 and 0.5 pM, between 0.01 and 0.25 pM, between 0.01 and 0.1 pM, between 0.1 and 100 pM, between 0.1 and 10 pM, between 0.1 and 5 pM, between 0.1 and 2.5 pM, between 0.1 and 1 pM, between 0.1 and 0.5 pM, between 0.1 and 0.25 pM, between 0.25 and 100 pM, between 0.25 and 10 pM, between 0.25 and 5 pM, between 0.25 and 2.5 pM, between 0.25 and 1 pM, between 0.25 and 0.5 pM, between 0.5 and 100 pM, between 0.5 and 10 pM, between 0.5 and 5 pM, between 0.5 and 2.5 pM, between 0.5 and 1 pM, between 1 and 100 pM, between 1 and 10 pM, between 1 and 5 pM, between 1 and 2.5 pM, between 2.5 and 100 pM, between 2.5 and 10 pM, between 2.5 and 5 pM, between 5 and 100 pM, between 5 and 10 pM, or such as wherein the Rep protein concentration is between 10 and 100 pM.

330. The method according to any one of the preceding claims, wherein the Rep protein concentration is 0.1 to 10 pM, such as 0.1 to 9 pM, such as 0.1 to 8 pM, such as 0.1 to 7 pM, such as 0.1 to 6 pM, such as 0.1 to 5 pM, such as 0.5 to 5 pM, such as 0.5 to 4 pM, such as 0.5 to 3 pM, such as 0.5 to 2 pM, such as 0.5 to 1 .5 pM.331 . The method according to any one of the preceding claims, wherein the Rep protein concentration is 1 pM.

332. The method according to any one of the preceding claims, wherein the Rep protein is purified Rep protein.

333. The method according to any one of the preceding claims, wherein the Rep protein is purified from Escherichia Coli.

334. The method according to any one of the preceding claims, wherein at least 80% of the assembled parvovirus capsids are filled by said method, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or wherein at least 99% of the assembled parvovirus capsids are filled by said method.

335. The method according to any one of the preceding claims, wherein 100% of the assembled parvovirus capsids are filled by said method.

336. The method according to any one of the preceding claims, wherein at least 80% of the assembled parvovirus capsids are filled with said transgene of interest by said method, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or wherein at least 99% of the assembled parvovirus capsids are filled by said method.

337. The method according to any one of the preceding claims, wherein 100% of the assembled parvovirus capsids are filled with said transgene of interest by said method.

338. The method according to any one of the preceding claims, wherein the method yields at least 80% filled parvovirus capsids, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% filled parvovirus capsids.

339. The method according to any one of the preceding claims, wherein the method yields 100% filled parvovirus capsids.

340. A fusion protein, such as a MBP-Rep78 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 35, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 35.341 . A fusion protein, such as a MBP-Rep78 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 35.

342. A fusion protein, such as a SUMO-Rep40 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 36, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91%, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 36.

343. A fusion protein, such as a SUMO-Rep40 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 36.

344. A fusion protein, such as a SUMO-Rep52 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 37, such as 81%, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91%, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 37.

345. A fusion protein, such as a SUMO-Rep52 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 37.

346. A fusion protein, such as a SUMO-Rep68 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 38, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 38.

347. A fusion protein, such as a SUMO-Rep68 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 38.

348. A fusion protein, such as a SUMO-Rep78 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 39, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 39.

349. A fusion protein, such as a SUMO-Rep78 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 39.

350. A fusion protein, such as a Smt3-Rep40 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 40, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 40.351 . A fusion protein, such as a Smt3-Rep40 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 40.

352. A fusion protein, such as a Smt3-Rep52 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 41 , such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 41 .

353. A fusion protein, such as a Smt3-Rep52 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 41 .

354. A fusion protein, such as a Smt3-Rep68 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 42, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 42.

355. A fusion protein, such as a Smt3-Rep68 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 42.

356. A fusion protein, such as a Smt3-Rep78 fusion protein, consisting of or comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 43, such as 81 %, such as 82%, such as 83%, such as 84%, such as 85%, such as 86%, such as 87%, such as 88%, such as 89%, such as 90%, such as 91 %, such as 92%, such as 93%, such as 94%, such as 95%, such as 96%, such as 97%, such as 98%, or such as 99% sequence identity to the amino acid sequence of SEQ ID NO: 43.

357. A fusion protein, such as a Smt3-Rep78 fusion protein, consisting of or comprising the amino acid sequence of SEQ ID NO: 43.

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