Production of recombinant aav

Abstract

To provide a method for producing a population of high titer recombinant adeno-associated virus (AAV) lacking prokaryotic sequences.SOLUTION: Culturing a human embryo cell strain in suspension, transfecting the human embryo cell strain with (a) nucleic acids sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acids sequences encoding AAVrep and AAVcap genes, and (c) a closed ended linear duplex rAAV vector nucleic acids comprising at least one inverted terminal repeats (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements, incubating the transfected human cell strain for about 40 to 400 hours, and optionally, B. lysing the transfected human cell line to purify the nucleic acid sequence encoding the rAAV, thereby producing the rAAV.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 962,911, filed January 17, 2020, the entire contents of which are incorporated by reference.

[0002] Field of the Disclosure The present disclosure relates to the production of recombinant adeno-associated virus (rAAV) virions that are devoid of prokaryotic sequences. [Background technology]

[0003] background Current methods for producing rAAV remain expensive despite years of research. Currently, approximately 10 5 A typical production rate of 10 genome copies (GC) / cell is 10 14GC / L is obtained (Kotin RM. Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011;20(R1):R2-R6. doi: 10.1093 / hmg / ddr141 (Non-Patent Document 1)). While this has been shown to be sufficient to support early clinical trials and may allow for commercial availability for small patient populations, the lack of scalability of this platform is a major limitation (Clement N, Grieger JC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev. 2016;3:16002. doi: 10.1038 / mtm.2016.2; Wright JF. Manufacturing and characterizing AAV-based vectors for use in clinical studies. Gene Ther. 2008;15(11):840-848. doi: 10.1038 / gt.2008.65). As can be imagined, successfully delivering three plasmids into a single cell is a relatively inefficient process. For large-scale manufacturing approaches, transient delivery of plasmids requires excessive amounts of DNA, increasing the overall cost of production and purification. Furthermore, transient delivery of rep / cap genes in the presence of helper genes can also contribute to product heterogeneity, including AAV vectors lacking a transgene. These "empty capsids" account for a large proportion of virus produced in transient transfection assays. Therefore, developing robust analytical quality control (QC) methods to ensure similarity between production lots is crucial. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kotin RM. Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011;20(R1):R2-R6. doi: 10.1093 / hmg / ddr141 [Non-patent document 2] Clement N, Grieger JC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev. 2016;3:16002. doi: 10.1038 / mtm.2016.2 [Non-patent document 3] Wright JF. manufacturing and characterizing AAV-based vectors for use in clinical studies. Gene Ther. 2008; 15(11):840-848. doi: 10.1038 / gt.2008.65 Summary of the Invention

[0005] Abstract Aspects of the present invention relate to the large-scale production of closed, linear recombinant adeno-associated virus (rAAV) vectors that lack prokaryotic sequences.

[0006] In one aspect, a method for producing recombinant adeno-associated virus (rAAV) is provided herein. Generally, this method includes the following steps: transfecting a closed-ended linear double-stranded rAAV vector nucleic acid into a host cell line in a culture medium, the closed-ended linear double-stranded rAAV vector nucleic acid includes (a) a nucleic acid sequence encoding a helper protein sufficient for rAAV replication, (b) a nucleic acid sequence encoding an AAV rep and AAV cap gene, and (c) at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements; for example, incubating the transfected host cell line for a sufficient time to produce rAAV; optionally, lysing the transfected host cell and isolating / purifying the rAAV from the culture medium. This method is suitable for producing high-titer rAAV. Therefore, the titer of the rAAV produced by this method can be higher than the titer of the rAAV produced using a host cell line transfected with a corresponding amount of plasmid DNA (pDNA) containing the same heterologous transgene. In certain embodiments, 1 x 10 6 The total amount of nucleic acid from (a), (b), and (c) per host cell is less than about 2 μg. In certain embodiments, the host cells are transfected using a transfection composition comprising (a), (b), and (c), and a polycationic polymer, wherein the ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:1 to about 3:1 (weight:weight). In some embodiments, the transfected host cells are lysed. In some other embodiments, the transfected cells are not lysed.

[0007] In certain embodiments, the titer of the rAAV produced by the methods of the present invention is higher than the titer of the rAAV produced using a host cell line transfected with a corresponding amount of plasmid DNA containing a heterologous transgene. For example, the rAAV titer produced by the methods of the present invention is at least 1.25-fold, e.g., 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or more, higher than the titer of the rAAV obtained using a corresponding amount of plasmid DNA.

[0008] In certain embodiments, a method for producing recombinant adeno-associated virus (rAAV) includes providing a vector encoding an AAV nucleic acid sequence or a closed, linear AAV nucleic acid sequence; culturing a human embryonic cell line in suspension; transfecting the human cell line with the vector encoding the AAV nucleic acid and a transfection composition or the closed, linear AAV nucleic acid sequence and a transfection composition; incubating the transfected human cell line for about 40 to 400 hours; and optionally lysing the transfected human cell line to purify the rAAV-encoding nucleic acid sequence, thereby producing rAAV. In some aspects of this embodiment, the transfected host cells are lysed. In some other embodiments, the transfected cells are not lysed.

[0009] In certain embodiments, the transfection composition includes (i) a vector encoding adenovirus helper proteins, (ii) a vector including an AAV rep gene and an AAV capsid (cap) protein gene, and (iii) a vector including an AAV inverted terminal repeat (ITR) sequence. In some embodiments, at least one of the vectors (i) to (iii) is included in a closed, linear AAV nucleic acid sequence. For example, vector (iii) is included in a closed-ended, linear, double-stranded vector nucleic acid including at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements.

[0010] In certain embodiments, the vector encoding the adenovirus helper proteins lacks adenovirus structural and replication genes. In certain embodiments, the AAV rep and capsid genes are derived from different serotypes. In certain embodiments, the AAV rep and capsid genes are derived from the same serotype. Examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV13, etc. In certain embodiments, the AAV rep gene is derived from a serotype selected from the group consisting of AAV2, 3, 8, 9, and 10. In certain embodiments, the AAV cap gene is derived from a serotype selected from the group consisting of AAV2, 3, 8, 9, and 10. For example, the AAV rep gene is the AAV2 rep gene, and the AAV capsid gene is the AAV8 capsid gene. In certain embodiments, the AAV inverted terminal repeat (ITR) sequences are adeno-associated virus 2 inverted terminal repeat (ITR) sequences. Virtually any other serotype combination can be used.

[0011] In certain embodiments, the rAAV particles have at least AAV ITRs from an AAV serotype selected from the group consisting of AAV1, 2, 3 (e.g., 3a, 3b), 4, 5, 6, 7, 8, 9, 10, 11, and 13. In certain embodiments, the AAV ITRs are from a serotype selected from the group consisting of AAV2, 3 (e.g., 3a, 3b), 8, 9, and 10. In certain embodiments, the AAV ITRs and the AAV cap gene are from different serotypes. In certain embodiments, the AAV ITRs and the AAV cap gene are from the same serotype. In some embodiments, the AAV ITRs are wild-type, mutant, or synthetic. In certain embodiments, the mutant ITRs include one or more amino acid substitutions, additions, and / or deletions. In some embodiments, the AAV ITRs are exemplary ITRs from US 7,790,154; US 8,361,457; US 8,784,799; US 9,447,433; US 9,169,494; or US 10,233,428.

[0012] In certain embodiments, the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line.

[0013] In certain embodiments, a suspension of a human embryonic cell line is cultured incrementally in increasing volumes prior to transfection. In certain embodiments, the volume of the culture is gradually increased from about 50 ml to about 100 liters. In certain embodiments, the culture medium for the gradual expansion of a human embryonic cell suspension from about 50 ml to about 10 liters contains an amino acid at a concentration of about 1 mM to about 20 mM. In certain embodiments, a culture medium having a volume of about 5 liters contains an amino acid at a concentration of about 10 mM. In certain embodiments, the amino acid is L-glutamine. In certain embodiments, a culture medium having a volume of about 50 liters contains at least about 1 mM to about 20 mM L-glutamine, at least about 0.01% to about 1% of a non-ionic surfactant polyol or surfactant, and at least about 0.001% to about 1% of an anti-foaming agent. In certain embodiments, the non-ionic surfactant polyol contains pluronic acid.

[0014] The cell density of the host cells was approximately 3.0 × 10 viable cells. 6 ~Approx. 1×10 8 For example, the cell density of the host cells can range from about 3.5 x 10 viable cells / ml. 7 ~Approx. 8.5×10 7 In certain embodiments, the cell density of the host cells can range from about 3 x 10 viable cells / ml. 6 ~about 6×10 6 For example, the cell density of the host cells can range from about 4.0 x 10 viable cells / ml. 6 ~about 6×10 6 In certain embodiments, the cell density of the host cells can be about 2.5 x 10 viable cells / ml. 7 In some other embodiments, the cell density of the host cells in the cell culture may be about 3 x 10 viable cells / ml. 7 It can be 1 / ml.

[0015] In certain embodiments, the cultured human embryonic cell line comprises about 3.0 x 10 viable cells. 6 ~Approx. 1×10 8 In one embodiment, the cultured human embryonic cell line comprises a cell density of 2.5 x 10 viable cells / ml. 7 In another embodiment, the cultured human embryonic cell line comprises 3 x 10 viable cells / ml. 7 In some embodiments, the cultured human embryonic cell line comprises about 3.5 x 10 viable cells / ml. 7 ~Approx. 8.5×10 7 In certain embodiments, the cultured human embryonic cell line comprises a cell density of about 4.0 x 10 viable cells / ml. 6 ~about 6×10 6 In certain embodiments, the human embryonic cell line comprises a cell density of about 3 x 10 viable cells / ml. 6 ~Approx. 5×10 6 The cells are transfected at a cell density of 1000 / ml with a vector encoding an AAV nucleic acid sequence and transfection composition, or with a closed linear AAV nucleic acid sequence and transfection composition.

[0016] In certain embodiments, the transfection composition comprises at least about 5% volume / volume (v / v) to about 50% v / v of culture medium. In certain embodiments, the transfection composition comprises at least about 5% volume / volume (v / v) to about 20% v / v of culture medium. In certain embodiments, the transfection composition comprises at least about 10% volume / volume (v / v) to about 20% v / v of culture medium. In some embodiments, the transfection composition comprises at least about 5% volume / volume (v / v) to about 10% v / v of culture medium. In certain embodiments, the transfection composition comprises about 1 liter to about 5 liters of culture medium. In certain embodiments, the nucleic acid sequence added to the transfection composition is at least 0.5 x 10 6 ~Approx. 5×10 6 Each cell contains about 0.1 μg to about 1 μg of Ad helper DNA, Rep / Cap DNA, or transgene.

[0017] In certain embodiments, the method further comprises (i) adding about 1 liter of medium to the transfected cells, and (ii) adding a cationic polymer at a ratio of about 1:1 polymer to DNA to about 3:1 polymer to DNA over a time course of about 1 to about 5 minutes. In certain embodiments, the cationic polymer is added at a ratio of 2.2:1 polymer to DNA over a time course of about 1 minute. In certain embodiments, the cationic polymer comprises fully hydrolyzed linear polyethyleneimine (PEI).

[0018] In certain embodiments, the temperature of the culture medium containing the host cells is raised to 37°C about 12-36 hours prior to transfection. For example, the temperature of the culture medium containing the human embryonic cell suspension is raised to 37°C about 12-36 hours prior to transfection.

[0019] In certain embodiments, the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM. In certain embodiments, the culture medium is subjected to air sparging at a flow rate of about 0.5 LPM. In certain embodiments, the culture medium is subjected to air sparging at a flow rate of about 1.5 LPM, 2 LPM, 5 LPM, 7 LPM, 10 LPM, 15 LPM, 20 LPM, 30 LPM, 40 LPM, 50 LPM, 60 LPM, 70 LPM, 80 LPM <90 LPM, or 100 LPM. In certain embodiments, the culture medium is maintained at a pH of at least about 7.0.

[0020] In certain embodiments, a method for producing high titer recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences comprises transfecting 1×10 mammalian cells with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acid sequences encoding rep and cap genes, and (c) at least one ITR sequence and a heterologous transgene operably linked to one or more regulatory elements, the method comprising transfecting 1×10 mammalian cells with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR sequence and a heterologous transgene operably linked to one or more regulatory elements, the heterologous transgene operably linked to at least one ITR sequence and ... 6wherein the total amount of nucleic acid transfected from (a), (b), and (c) per cell is less than 1 μg; culturing the transfected cells for at least 24 hours, e.g., at least 40 hours; optionally lysing the transfected cells and purifying the rAAV vector particles produced, wherein the titer of rAAV is at least 9.3 x 10 13 Vector genomes / 3.0×10 9 In some aspects of this embodiment, the transfected host cells are lysed. In some other embodiments, the transfected cells are not lysed.

[0021] In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 10 ~ at least 1×10 16 vector genomes / 1.0×10 8 ~1×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 11 vector genomes / 1.0×10 9 ~2×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 12 Vector genomes / 2.0×10 9 ~3×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 13 Vector genomes / 2.0×10 9 ~2×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 14 Vector genomes / 2.0×10 9 ~4×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 2 x 10 14 Vector genomes / 3.0×109 ~5×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 3 x 10 14 Vector genomes / 4.0×10 9 ~5×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 4 x 10 14 vector genomes / 5.0×10 9 ~5×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 5 x 10 14 Vector genomes / 6.0×10 9 ~5×10 11 In certain embodiments, the titer of the rAAV vector particles is at least 1.25 x 10 14 Vector genomes / 4.0×10 9 transfected live cells.

[0022] In certain embodiments, the titer of the rAAV vector particles is at least 2 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 2.5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 3 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 3.5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 4 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 4.5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 5.5 x 10 11In certain embodiments, the titer of the rAAV vector particles is at least 6 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 6.5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 7 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 7.5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 8 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 8.5 x 10 11 In certain embodiments, the titer of the rAAV vector particles is at least 9 x 10 vp / ml. 11 In certain embodiments, the titer of the rAAV vector particles is at least 9.5 x 10 vp / ml. 11 In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 vp / ml. 12 In certain embodiments, the titer of the rAAV vector particles is at least 5 x 10 12 vp / ml.

[0023] In certain embodiments of the invention, the titer of rAAV vector particles obtained using closed linear (c1DNA) is at least 1 e 11 In some embodiments, the titer of the rAAV vector particles is at least 2e 11 In some embodiments, the titer of the rAAV vector particles is at least 3 e 11 In certain embodiments, the titer of the rAAV vector particles is at least 4e 11 In certain embodiments, the titer of the rAAV vector particles is at least 5e 11 In various embodiments, the titer of the rAAV vector particles is at least 6e 11In some embodiments, the titer of the rAAV vector particles is at least 7e 11 In certain embodiments, the titer of the rAAV particles is at least 8e 11 In some embodiments, the titer of the rAAV particles is at least 8.5 e 11 In other embodiments, the titer of the rAAV particles is at least 9 e 11 In yet another embodiment, the titer of the rAAV particles is at least 9.5 e 11 In certain embodiments, the titer of the rAAV particles is at least 1 e 12 vg / ml.

[0024] In some embodiments, rAAV vector particles obtained using closed linear (cDNA) are about 2 to about 3 times more abundant than those obtained using plasmid DNA (pDNA). In other embodiments, rAAV vector particles obtained using closed linear (cDNA) are about 4 to about 5 times more abundant than those obtained using plasmid DNA (pDNA). In other embodiments, rAAV vector particles obtained using closed linear (cDNA) are about 6 to about 8 times more abundant than those obtained using plasmid DNA (pDNA). In various embodiments, rAAV vector particles obtained using closed linear (cDNA) are about 9 to about 15 times more abundant than those obtained using plasmid DNA (pDNA).

[0025] Certain embodiments of the methods described herein involve the use of a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) a nucleic acid sequence encoding a helper protein, (b) a nucleic acid sequence encoding the rep and cap genes, and (c) a heterologous transgene operably linked to at least one ITR and one or more regulatory elements. The ratio of (a) the nucleic acid sequence encoding the helper protein to (b) the nucleic acid sequence encoding the rep and cap genes to (c) the heterologous transgene operably linked to at least one ITR and one or more regulatory elements [(a):(b):(c)] can be optimized for the specific nucleic acid used. For example, the ratio of (a):(b):(c) can be about 0.5-1.75: about 0.75-2.25: about 0.5-1.75 (weight:weight:weight). In certain embodiments, the ratio of (a):(b):(c) is about 0.75-1.5: about 1-1.75: about 0.75-1.25 (wt:wt:wt).

[0026] In certain embodiments, the ratio of (a) a nucleic acid sequence encoding a helper protein to (b) a nucleic acid sequence encoding rep and cap genes to (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements [(a):(b):(c)] is about 1:about 1 to 1.6:about 1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 0.5:1:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 0.5:1:0.5 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 0.75:1:0.75 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 0.5:1:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 0.5:1:0.75 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 0.5:1.5:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.5:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.6:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.75:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.8:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.85:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.90:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:1.95:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1:2:1 (wt:wt:wt). In certain embodiments, the ratio of (a):(b):(c) is about 1.4:about 1.5:about 1 (wt:wt:wt).

[0027] In certain embodiments, host cells are transfected with a transfection composition comprising (a) a nucleic acid sequence encoding a helper protein, (b) a nucleic acid sequence encoding the rep and cap genes, (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, and (d) a polycationic polymer. The cationic polymer can be any synthetic or natural polymer with at least two positive charges per molecule and sufficient charge density and molecular size to bind to nucleic acids under physiological conditions. In certain embodiments, the polycationic polymer contains one or more amine residues, such as polyethyleneimine (PEI), or polyamino acids such as polyornithine, polyarginine, and polylysine. In a preferred embodiment, the polycationic polymer is PEI.

[0028] Polycationic polymers can be any synthetic or natural polymer with at least two positive charges per molecule and sufficient charge density and molecular size to bind to nucleic acids under transfection conditions (i.e., pH and salt conditions encountered in cell culture). Suitable cationic polymers include, for example, polyethyleneimine (PEI), polyallylamine, polyvinylamine, polyvinylpyridine, aminoacetalized poly(vinyl alcohol), acrylic or methacrylic acid polymers with one or more amine residues (e.g., poly(N,N-dimethylaminoethyl methacrylate)), polyamino acids such as polyornithine, polyarginine, and polylysine, protamine, cationic polysaccharides such as chitosan, DEAE-cellulose, and DEAE-dextran, and polyamidoamine dendrimers (cationic dendrimers), as well as copolymers and blends thereof.

[0029] Polycationic polymers can be either linear or branched, either homopolymers or copolymers, and, if they contain amino acids, can have either an L- or D-configuration, or any combination of these features. Preferably, the cationic polymer molecule has sufficient flexibility to allow it to form compact complexes with one or more nucleic acid molecules.

[0030] The molecular weight of the polycationic polymer can be varied depending on the properties of one or more nucleic acids. Thus, in some embodiments, the polycationic polymer has a molecular weight of about 5,000 to about 100,000 daltons, more preferably about 5,000 to about 50,000 daltons, and most preferably about 10,000 to about 35,000 daltons.

[0031] In certain embodiments, the polycationic polymer is polyethyleneimine (PEI). For example, the polycationic polymer is linear polyethyleneimine. In certain embodiments, the polycationic polymer is fully hydrolyzed polyethyleneimine.

[0032] In some embodiments, the polycationic polymer is a stable cationic polymer.

[0033] The ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) can be varied for optimal transfection. For example, the ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) can be about 1.5:1 to about 2.75:1. In certain embodiments, the ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) can be about 1.9:1 to about 2.6:1. In certain embodiments, the ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) can be about 1:1.5 to about 1:2.75. For example, the ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) can be about 1:1.9 to about 1:2.6.

[0034] The polycationic polymer is present in the transfection composition in an amount effective to complex with the nucleic acid from (a), (b), and (c) to form a complex. In certain embodiments, the relative amounts of polycationic polymer and nucleic acid from (a), (b), and (c) can be represented by the number of nitrogen atoms in the polycationic polymer divided by the number of phosphorus atoms in the nucleic acid (N / P ratio). In certain embodiments, the polycationic polymer and nucleic acid from (a), (b), and (c) are present at an N / P ratio of about 2 to about 15, more preferably about 3 to about 12, and most preferably about 4 to about 9.

[0035] In certain embodiments, steps (a), (b), and (c) are transfected using a transfection composition comprising (a), (b), and (c), and a stable cationic polymer, and the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1.5:1. In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 0.5:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 0.75:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1.75:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 2:1 (weight:weight), about 2.2:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 2.5:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 3:1 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:0.75 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:0.5 (weight:weight). In certain embodiments, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:0.25 (weight:weight).

[0036] In certain embodiments, each of (a), (b), and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules. In certain embodiments, the transfected nucleic acids (a), (b), and (c) are synthetic nucleic acids and do not have prokaryotic DNA modifications. In certain embodiments, the transfected (a), (b), and (c) are synthetic nucleic acids and do not have eukaryotic or prokaryotic DNA modifications. In certain embodiments, the nucleic acid packaged within purified recombinant AAV (rAAV) is devoid of prokaryotic and eukaryotic DNA sequences. In one embodiment, non-AAV vector DNA comprises less than 10% of the total DNA within the rAAV particle.

[0037] In certain embodiments, a method for producing a population of purified recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences comprises the steps of transfecting a mammalian cell line suspended in culture medium with a transfection composition, the transfection composition comprising: (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acid sequences encoding rep and cap genes, and (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, and (d) a stable cationic polymer, wherein the ratio of stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is at least 1.5:1; culturing the transfected cell line for at least 24 hours, e.g., at least 40 hours; lysing the transfected cell line of step (ii); and purifying the rAAV, wherein the purified virus is at least 2×10 4 In some aspects of this embodiment, the transfected host cells are optionally lysed. In some embodiments, the purified recombinant AAV (rAAV) produced has a particle to infectivity ratio of less than 1.5 x 10 4In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 1 x 10 vg / TCID. 4 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 9×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 8×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 7×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 6×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 5×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 4×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 3×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 2×10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 1 x 10 vg / TCID. 3 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 9×10 vg / TCID. 2 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 8×10 vg / TCID. 2 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 7×10 vg / TCID. 2 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 6×10 vg / TCID.2 In some embodiments, the purified recombinant AAV (rAAV) has a particle-to-infectivity ratio of less than 5×10 vg / TCID. 2 have a particle-to-infectivity ratio of less than vg / TCID50.

[0038] In a particular embodiment of any one of these aspects, the infectious particle titer is at least 1 x 10 4 In certain embodiments, the infectious particle titer is at least 1.5 x 10 vg / TCID50. 4 In certain embodiments, the infectious particle titer is at least 2 x 10 vg / TCID50. 4 In certain embodiments, the infectious particle titer is at least 2.5 x 10 vg / TCID50. 4 In certain embodiments, the infectious particle titer is at least 3 x 10 vg / TCID50. 4 In certain embodiments, the infectious particle titer is at least 3.5 x 10 vg / TCID50. 4 In certain embodiments, the infectious particle titer is at least 4 x 10 vg / TCID50. 4 In certain embodiments, the infectious particle titer is at least 4.5 x 10 vg / TCID50. 4 In certain embodiments, the purified virus is at least 5×10 vg / TCID50. 4 It has a particle-to-infectivity ratio of vg / TCID50.

[0039] In some embodiments, the method of producing recombinant AAV uses a transient transfection method. In some embodiments, the method of producing recombinant AAV uses a stable transfection method. In various embodiments, the transfection is performed in suspension.

[0040] An embodiment of the method described herein includes incubating the inoculated cell culture medium, e.g., the transfected host cells, for a period during which rAAV is produced. For example, the inoculated cell culture medium, e.g., the transfected host cells, can be incubated for a period of at least 24 hours. For example, the transfected host cells can be incubated for a period of at least 30 hours. In certain embodiments, the inoculated cell culture medium, e.g., transfected host cells, is incubated for 30 to 100 hours, or 30 to 150 hours, or 30 to 200 hours, or 40 to 100 hours, or 40 to 150 hours, or 40 to 200 hours, or 40 to 300 hours, or 40 to 350 hours, or 40 to 400 hours, or 40 to 450 hours, or 40 to 500 hours, or 40 to 550 hours, or 40 to 600 hours, or 40 to 650 hours, or 40 to 700 hours, or 40 to 750 hours, or 40 to 800 hours, or 40 to 850 hours, or 40 to 900 hours, or 40 to 950 hours, or 40 to 1000 hours. In certain embodiments, the inoculated cell culture medium, e.g., transfected cells, is cultured for about 40 to 400 hours. In certain embodiments, the transfected cells are cultured for about 40 to 100 hours, or about 40 to 150 hours, or about 40 to 200 hours, or about 40 to 250 hours, or about 40 to 300 hours, or about 40 to 350 hours, or about 40 to 400 hours, or about 40 to 450 hours, or about 40 to 450 hours, or about 40 to 500 hours, or about 40 to 550 hours, or about 40 to 600 hours, or about 40 to 650 hours, or about 40 to 700 hours, or about 40 to 750 hours, or about 40 to 800 hours, or about 40 to 850 hours, or about 40 to 900 hours, or about 40 to 950 hours, or about 40 to 1000 hours.In certain embodiments, the inoculated cell culture medium, e.g., transfected cells, are cultured for at least 24 hours, or at least 30 hours, or at least 40 hours, or at least 45 hours, or at least 50 hours, or at least 55 hours, or at least 60 hours, or at least 65 hours, or at least 70 hours, or at least 72 hours, or at least 75 hours. In certain embodiments, the inoculated cell culture medium, e.g., transfected cells, are cultured for 1000 hours or less, or 950 hours or less, or 900 hours or less, or 850 hours or less, or 800 hours or less, or 750 hours or less, or 700 hours or less, or 650 hours or less, or 600 hours or less, or 550 hours or less, or 500 hours or less, or 450 hours or less, or 400 hours or less, or 350 hours or less, or 300 hours or less, or 250 hours or less, or 200 hours or less, or 150 hours or less, or 100 hours or less.

[0041] In certain embodiments, the mammalian cell line is a suspension cell or cell line, i.e., a non-adherent cell or cell line, and the cells are transfected in suspension. In certain embodiments, the cell line is derived from the human embryonic kidney 293 cell line (HEK293). In certain embodiments, the human embryonic kidney cells lack SV40 antigens or other transforming antigens. In certain embodiments, the mammalian cell line is a suspension-adapted serum-free cell line. In certain embodiments, the cell line is derived from primary blood cells, such as lymphocytes, monocytes, macrophages, granulocytes, dendritic cells, and erythrocytes. In certain embodiments, the cell line is derived from a cell biopsy, including, for example, lymph node cells, bone marrow cells, and umbilical cord blood cells. In certain embodiments, the cell line is derived from circulating tumor cells. In certain embodiments, the cell line is derived from a blood cell line, such as Jurkat and Molt4 T cell lines, U937 and THP promonocytic cell lines, and B cell hybridomas. In certain embodiments, the cell line is derived from a stem cell. In certain embodiments, the cell line used for the production of recombinant AAV is a stable cell line.

[0042] In certain embodiments, a suspension of a mammalian cell line is progressively cultured under increasing volumes of culture medium prior to transfection.

[0043] The methods disclosed herein are scalable and can be applied to the efficient and scalable production of rAAV. In other words, the methods described herein can be used in volumes ranging from a few milliliters to thousands of liters. Thus, the described methods can be used for industrial-scale production of therapeutic rAAV compositions. In certain embodiments, the volume of a cell culture containing host cells can be at least about 50 liters. For example, the volume of the cell culture can be from about 50 liters to about 4000 liters. In certain embodiments, the volume of the cell culture can be from about 50 liters to about 2000 liters. For example, the volume of the cell culture can be from about 50 liters to about 250 liters. In another non-limiting example, the volume of the cell culture can be from about 50 liters to about 100 liters.

[0044] The volume of the cell culture containing the host cells can be increased before transfection. For example, the volume of the cell culture can be increased from about 10-20, 30, 40, or 50 ml to about 4,000 liters. In certain embodiments, the volume of the cell culture can be increased from about 10-20, 30, 40, or 50 ml to about 2,000 liters. For example, the volume of the cell culture can be increased from about 10-20, 30, 40, or 50 ml to about 250 liters. In another non-limiting example, the volume of the cell culture can be increased from about 10-20, 30, 40, or 50 ml to about 50 liters or about 100 liters. In certain embodiments, the volume of the cell culture can be increased from about 100 liters. In certain embodiments, the volume of the cell culture can be increased from about 50 ml to about 50 liters. In certain embodiments, the cell culture volume may be increased from a volume of about 50 ml to a volume of about 10 liters.

[0045] In certain embodiments, the culture volume is gradually increased from about 50 ml to about 4000 liters. In certain embodiments, the culture volume is gradually increased from about 50 ml to about 2000 liters. In certain embodiments, the culture volume is gradually increased from about 10-20, 30, 40, or 50 ml to about 250 liters. In certain embodiments, the culture volume is gradually increased from about 10-20, 30, 40, or 50 ml to about 100 liters. In certain embodiments, the culture volume is gradually increased from about 10-20, 30, 40, or 50 ml to about 50 liters. In certain embodiments, the culture medium for the gradual expansion of a human embryonic cell suspension from about 50 ml to about 50 liters comprises an amino acid at a concentration of about 1 mM to about 20 mM. In certain embodiments, a culture medium having a volume of about 5 liters contains an amino acid at a concentration of about 10 mM. In certain embodiments, the amino acid is L-glutamine.

[0046] In certain embodiments, the packaged nucleic acid of the rAAV virion lacks prokaryotic DNA sequences.

[0047] Certain embodiments include nucleic acid sequences encoding helper proteins sufficient for rAAV replication. Helper proteins sufficient for rAAV replication have been widely studied, and many adenovirus genes encoding helper protein functions have become known. For example, proteins encoded by early adenovirus gene regions E1A (e.g., present in HEK293 cells), E2A, E4Orf6, VAI RNA, and optionally VAII RNA, and optionally E1B (also present in HEK293 cells), are believed to be involved in the rAAV replication process. Thus, in certain embodiments, nucleic acid sequences encoding helper proteins sufficient for rAAV replication include nucleotide sequences encoding adenovirus (Ad) helper proteins. For example, nucleic acid sequences encoding helper proteins sufficient for rAAV replication include nucleotide sequences encoding adenovirus helper proteins E2A and / or E4.

[0048] In certain embodiments, (a) the nucleic acid sequence encoding a helper protein sufficient for rAAV replication is an adenovirus (Ad) helper comprising nucleic acids encoding adenovirus helper proteins E2A and E4.

[0049] In certain embodiments, the total amount of DNA from (a), (b), and (c) is about 1 to about 50 μg. In certain embodiments, the total amount of DNA from (a), (b), and (c) is about 1 to about 20 μg. In certain embodiments, the total amount of DNA from (a), (b), and (c) is about 1 to about 10 μg. In certain embodiments, the total amount of DNA from (a), (b), and (c) is about 1 to about 8 μg. In certain embodiments, the total amount of DNA from (a), (b), and (c) is about 1 to about 6 μg. In certain embodiments, the total amount of DNA from (a), (b), and (c) is about 1 to about 3 μg. In certain embodiments, the total amount of DNA from (a), (b), and (c) is, optionally, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, or 1.8 μg. In a particular embodiment, the total amount of DNA from (a), (b), and (c) is 0.75 μg.

[0050] In certain embodiments, the total amount of nucleic acid from a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication; (b) nucleic acid sequences encoding the AAV rep and AAV cap genes; and (c) a heterologous transgene operably linked to at least one inverted terminal repeat (ITR) sequence and one or more regulatory elements, used to inoculate a cell culture, e.g., to transfect a host cell, is 1×10 6 For example, the total amount of nucleic acid from (a), (b), and (c) is less than about 2 μg per cell. 6 In certain embodiments, the total amount of nucleic acid from (a), (b), and (c) is less than about 1×10 6 Approximately less than 1 μg per cell, or 1 x 10 6Less than about 0.75 μg per cell.

[0051] In certain embodiments, the total amount of nucleic acid from (a), (b), and (c) is 1×10 6 For example, the total amount of nucleic acid from (a), (b), and (c) is 1 x 10 6 In certain embodiments, the total amount of nucleic acid from (a), (b), and (c) is at least 1×10 6 Approximately 0.25 μg to 1 × 10 cells 6 For example, the total amount of nucleic acid from (a), (b), and (c) is 1×10 6 Approximately 0.5 μg to 1 × 10 per cell 6 In certain embodiments, the total amount of nucleic acid from (a), (b), and (c) is about 1×10 6 Approximately 0.5 μg to 1 × 10 per cell 6 Approximately 0.75 μg per cell.

[0052] In certain embodiments, the infectious particle titer is at least 3 x 10 9 In certain embodiments, the infectious particle titer is at least 1 x 10 5 TCID50 / ml (Median Tissue Culture Infectious Dose) ~ Approx. 1×10 11 In certain embodiments, the infectious particle titer is at least 2 x 10 5 In certain embodiments, the infectious particle titer is at least 5 x 10 5 In certain embodiments, the infectious particle titer is at least 7.5 x 10 5 In certain embodiments, the infectious particle titer is at least 8 x 10 5 In certain embodiments, the infectious particle titer is at least 8.5 x 10 5In certain embodiments, the infectious particle titer is at least 9 x 10 5 In certain embodiments, the infectious particle titer is at least 9.5 x 10 5 In certain embodiments, the infectious particle titer is at least 9.9 x 10 5 In certain embodiments, the infectious particle titer is at least 1 x 10 6 In certain embodiments, the infectious particle titer is at least 1 x 10 6 In certain embodiments, the infectious particle titer is at least 2 x 10 6 In certain embodiments, the infectious particle titer is at least 5 x 10 6 In certain embodiments, the infectious particle titer is at least 7.5 x 10 6 In certain embodiments, the infectious particle titer is at least 8 x 10 6 In certain embodiments, the infectious particle titer is at least 8.5 x 10 6 In certain embodiments, the infectious particle titer is at least 9 x 10 6 In certain embodiments, the infectious particle titer is at least 9.5 x 10 6 In certain embodiments, the infectious particle titer is at least 9.9 x 10 6 In certain embodiments, the infectious particle titer is at least 1 x 10 7 In certain embodiments, the infectious particle titer is at least 2 x 10 7 In certain embodiments, the infectious particle titer is at least 5 x 10 7 In certain embodiments, the infectious particle titer is at least 7.5 x 10 7 In certain embodiments, the infectious particle titer is at least 8 x 10 7In certain embodiments, the infectious particle titer is at least 9 x 10 7 In certain embodiments, the infectious particle titer is at least 9.9 x 10 7 In certain embodiments, the infectious particle titer is at least 1 x 10 8 In certain embodiments, the infectious particle titer is at least 2.5 x 10 8 In certain embodiments, the infectious particle titer is at least 5 x 10 8 In certain embodiments, the infectious particle titer is at least 7.5 x 10 8 In certain embodiments, the infectious particle titer is at least 8 x 10 8 In certain embodiments, the infectious particle titer is at least 8.5 x 10 8 In certain embodiments, the infectious particle titer is at least 9 x 10 8 In certain embodiments, the infectious particle titer is at least 9.5 x 10 8 In certain embodiments, the infectious particle titer is at least 9.9 x 10 8 In certain embodiments, the infectious particle titer is at least 0.5 x 10 9 In certain embodiments, the infectious particle titer is at least 1 x 10 9 In certain embodiments, the infectious particle titer is at least 1.5 x 10 9 In certain embodiments, the infectious particle titer is at least 2 x 10 9 In certain embodiments, the infectious particle titer is at least 2.5 x 10 9 In certain embodiments, the infectious particle titer is at least 3 x 10 9 In certain embodiments, the infectious particle titer is at least 3.5 x 10 9In certain embodiments, the infectious particle titer is at least 4 x 10 9 In certain embodiments, the infectious particle titer is at least 4.5 x 10 9 In certain embodiments, the infectious particle titer is at least 5 x 10 9 In certain embodiments, the infectious particle titer is at least 5.5 x 10 9 In certain embodiments, the infectious particle titer is at least 6 x 10 9 In certain embodiments, the infectious particle titer is at least 6.5 x 10 9 In certain embodiments, the infectious particle titer is at least 7 x 10 9 In certain embodiments, the infectious particle titer is at least 7.5 x 10 9 In certain embodiments, the infectious particle titer is at least 8 x 10 9 In certain embodiments, the infectious particle titer is at least 8.5 x 10 9 In certain embodiments, the infectious particle titer is at least 9 x 10 9 In certain embodiments, the infectious particle titer is at least 9.5 x 10 9 In certain embodiments, the infectious particle titer is at least 9.9 x 10 9 In certain embodiments, the infectious particle titer is at least 1 x 10 10 In certain embodiments, the infectious particle titer is at least 2 x 10 10 In certain embodiments, the infectious particle titer is at least 5 x 10 10 In certain embodiments, the infectious particle titer is at least 7.5 x 10 10 In certain embodiments, the infectious particle titer is at least 8 x 10 10In certain embodiments, the infectious particle titer is at least 8.5 x 10 10 In certain embodiments, the infectious particle titer is at least 9 x 10 10 In certain embodiments, the infectious particle titer is at least 9.5 x 10 10 In some embodiments, the infectious particle titer is preferably normalized to vg / ml. In some embodiments, the infectious particle titer is at least 10 11 TCID50 / ml.

[0053] In certain embodiments, a method for large-scale production of recombinant adeno-associated virus (rAAV) comprises providing a vector encoding an AAV nucleic acid sequence or a closed linear AAV nucleic acid sequence, culturing a human embryonic cell line in suspension, transfecting the human cell line with the vector encoding the AAV nucleic acid sequence and a transfection composition or the closed linear AAV nucleic acid sequence and a transfection composition, incubating the transfected human cell line for about 30 to 250 hours, lysing the transfected human cell line, and purifying the nucleic acid sequence encoding the rAAV, thereby providing large-scale production of rAAV. In certain embodiments, the transfected cells are incubated for 30 to 100 hours, or 30 to 150 hours, or 30 to 200 hours, or 40 to 100 hours, or 40 to 150 hours, or 40 to 200 hours, or 40 to 300 hours, or 40 to 350 hours, or 40 to 400 hours, or 40 to 450 hours, or 40 to 500 hours, or 40 to 550 hours, or 40 to 600 hours, or 40 to 650 hours, or 40 to 700 hours, or 40 to 750 hours, or 40 to 800 hours, or 40 to 850 hours, or 40 to 900 hours, or 40 to 950 hours, or 40 to 1000 hours.

[0054] In certain embodiments, the AAV Rep gene and the AAV Cap gene are from the same AAV serotype. In certain embodiments, the AAV Rep gene and the AAV Cap gene are from different AAV serotypes.

[0055] In certain embodiments, the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line. The human embryonic cell line suspension is gradually cultured in increasing volumes prior to transfection. In certain aspects, the culture volume is gradually increased from about 50 ml to about 100 liters. In certain embodiments, the culture medium for the gradual expansion of the human embryonic cell suspension contains L-glutamine at a concentration of about 1 mM to about 20 mM in a culture medium volume of about 50 ml to about 10 liters. In certain embodiments, a culture medium having a volume of about 5 liters contains L-glutamine at a concentration of about 10 mM. In certain embodiments, a culture medium having a volume of about 50 liters contains at least about 1 mM to about 20 mM L-glutamine, at least about 0.01% to about 1% pluronic acid, and at least about 0.001% to about 1% antifoaming agent.

[0056] In certain embodiments, the cultured human embryonic cell line comprises about 3.0 x 10 viable cells. 6 ~Approx. 1×10 8 In certain aspects, the cultured human embryonic cell line comprises a cell density of about 4.0 x 10 viable cells / ml. 6 ~about 6×10 6 In certain embodiments, the human embryonic cell line comprises a cell density of about 3 x 10 viable cells / ml. 6 ~Approx. 5×10 6 pieces / ml 3 At a cell density of 1000 ng / mL, the cells are transfected with a vector encoding an AAV nucleic acid sequence and a transfection composition, or with a closed linear AAV nucleic acid sequence and a transfection composition.

[0057] In certain embodiments, the transfection composition comprises (i) a vector encoding an adenovirus helper protein, (ii) a vector comprising an AAV rep gene and an AAV capsid (cap) protein gene, and (iii) a vector comprising an AAV inverted terminal repeat (ITR) sequence. In certain embodiments, the vector encoding the adenovirus helper protein lacks adenovirus structural and replication genes. In certain aspects, the AAV rep and capsid genes are of different serotypes or the same serotype. In certain aspects, the AAV rep gene is the AAV2 rep gene, and the AAV capsid gene is the AAV8 capsid gene. In certain aspects, the AAV inverted terminal repeat (ITR) sequence is the adeno-associated virus 2 inverted terminal repeat (ITR) sequence. In certain embodiments, the nucleic acid sequence added to the transfection composition is 0.5×10 6 ~Approx. 5×10 6 The transfection comprises about 0.1 μg to about 1 μg of Ad helper DNA, Rep / Cap DNA, or transgene per cell. In some embodiments, transfection is carried out over a time course of about 10 minutes to about 60 minutes. In certain embodiments, transfection is carried out over a time course of about 10 minutes to about 120 minutes.

[0058] In certain embodiments, the cell density at the time of transfection is about 2.0 x 10 viable cells. 4 ~Approx. 1.0×10 8 In certain embodiments, the cell density at the time of transfection is about 5.0 x 10 viable cells / ml. 4 ~Approx. 5.0×10 7 In certain embodiments, the cell density at the time of transfection is about 1.0 x 10 viable cells / ml. 5 ~Approx. 1×10 7 In certain embodiments, the cell density at the time of transfection is about 5.0 x 10 viable cells / ml. 5 ~Approx. 1×10 7 In certain embodiments, the cell density at the time of transfection is about 2.0 x 10 viable cells / ml. 5 ~Approx. 9×106 In certain embodiments, the cell density at the time of transfection is about 1.0 x 10 viable cells / ml. 6 ~Approx. 7.5×10 6 In certain embodiments, the cell density at the time of transfection is about 1.0 x 10 viable cells / ml. 6 ~Approx. 7×10 6 In certain embodiments, the cell density at the time of transfection is about 1.0 x 10 viable cells / ml. 6 ~Approx. 5×10 6 In certain embodiments, the cell density at the time of transfection is about 1.0 x 10 viable cells / ml. 6 ~Approx. 4×10 6 pieces / ml.

[0059] Certain embodiments include transfection compositions for transfecting host cells. Generally, the transfection composition comprises: (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication; (b) a nucleic acid sequence encoding the AAV rep and AAV cap genes; (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements; and (d) a polycationic polymer. In addition to nucleic acids (a), (b), and (c), the transfection composition may also comprise cell culture medium, i.e., the medium used for the host cells. The transfection composition may have a volume of about 5% to about 20% (volume / volume) of the volume of the host cell line culture. For example, the host cell line is transfected with a transfection composition in a volume of about 7.5% to about 15% (volume / volume) of the volume of the host cell line culture.

[0060] In certain embodiments, the transfection composition comprises at least about 5% volume / volume (v / v) to about 20% v / v of culture medium. In certain embodiments, the transfection composition comprises about 1 liter to about 5 liters of medium. After the cells are transfected, about 1 liter of medium is added to the transfected cells. In certain embodiments, fully hydrolyzed linear polyethyleneimine (PEI) is added at a ratio of about 1:1 (PEI:DNA) to about 3:1 (PEI:DNA) over a time course of about 1 to about 5 minutes. In certain embodiments, fully hydrolyzed linear polyethyleneimine (PEI) is added at a ratio of 2.2:1 (PEI:DNA) over a time course of about 1 minute. In certain aspects, the suspension of transfected cells is incubated for about 1 to 20 minutes before being transferred to a large-volume bioreactor. In certain embodiments, the transfected cell suspension is incubated for about 3 hours and quenched with 10% (v / v) volume of chemically defined serum-free medium supplemented with about 10 mM L-glutamine. In certain embodiments, the temperature of the culture medium containing the human embryonic cell suspension is raised to 37°C about 12 to 36 hours prior to transfection. In certain embodiments, the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM. In certain embodiments, the culture medium is subjected to air sparging at a flow rate of about 0.5 LPM. In certain embodiments, the culture medium is maintained at a pH of at least about 7.0.

[0061] In certain embodiments, the recombinant adeno-associated virus (rAAV) comprises a protelomerase target sequence. In certain aspects, the protelomerase target sequence comprises a double-stranded palindromic sequence at least 10 base pairs in length. In certain embodiments, the rAAV comprises a transgene.

[0062] In certain embodiments, the pharmaceutical composition comprises a closed, linear recombinant adeno-associated virus (rAAV). In certain embodiments, the rAAV comprises a transgene.

[0063] In certain embodiments, the rAAV particles have AAV capsid genes from AAV serotypes including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16. In certain embodiments, the rAAV particles have AAV rep genes from AAV serotypes including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16. In some embodiments, but not limited to, the rAAV particle is an exemplary rAAV described in U.S. Patent No. 10,550,405, published International Application No. WO2018170310A1, U.S. Patent No. 7,892,809, U.S. Patent No. 6,491,907, or U.S. Patent No. 7,172,893. In certain embodiments, the rAAV is a hybrid AAV comprising an ITR from a specific AAV serotype and a capsid from a different AAV serotype. In some embodiments, the rAAV can comprise an rAAV virion. In certain embodiments, the rAAV capsid comprises one or more amino acid substitutions, additions, and / or deletions, for example, the capsid comprises an inserted peptide for targeting. [Brief explanation of the drawings]

[0064] This patent or application contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0065] [Figure 1] Figure 1 is a graph showing a comparison of vector genome titers in cell lysates, expressed as vg / mL yield, for the preferred transfection conditions using a plasmid-based (AskBio) system (N=2) and the clDNA system (N=5). Plots represent the mean + / - 1 standard deviation. [Figure 2]Figure 2 is a predictive profiler plot showing that both μg cDNA / 1E6 cells and PEI:DNA ratio have a statistically significant effect on Vg titer. p≦0.0066 [Figure 3] FIG. 3 is a contour plot showing the effect of clDNA and PEI:DNA ratio on vector genome titer in cell lysates, expressed in vg / mL. [Figure 4] FIG. 4 is a plot showing the effect of clDNA and PEI:DNA ratio on viral titer, expressed as vp / mL. [Figure 5] FIG. 5 is a predictive profiler plot showing that the PEI:DNA ratio, but not the amount of clDNA, has a statistically significant effect on the yield of AAHrh10 CYP titer expressed in vp / mL. [Figure 6] FIG. 6 is a predictive profiler plot showing that both μg clDNA / 1E6 cells and PEI:DNA ratio have a statistically significant effect on AAV8 GAA titer yield expressed in vp / ml. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description AAV is a protein shell that surrounds and protects a small, single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the Parvoviridae family and depends on coinfection with other viruses, primarily adenovirus, for replication. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in multiple species. Its single-stranded genome contains three genes: Rep (replication), Cap (capsid), and aap (assembly). These three genes generate at least nine gene products through the use of three promoters, alternative translation start sites, and alternative splicing. These coding sequences are flanked by inverted terminal repeats (ITRs), which are required for genome replication and packaging. The Rep gene encodes proteins (Rep78, Rep68, Rep52, and Rep40) required for viral genome replication and packaging, while Cap expression produces viral capsid proteins (VP; VP1 / VP2 / VP3) that form the outer capsid shell that protects the viral genome and actively participates in cell binding and internalization (Samulski RJ, Muzyczka N. AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol. 2014;1(1):427-451. doi: 10.1146 / annurev-virology-031413-085355). The viral envelope is composed of 60 proteins arranged in an icosahedral structure with capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3). The aap gene encodes an assembly-activating protein (AAP) in an alternative reading frame that overlaps with the cap gene. This nucleoprotein is thought to provide a scaffolding function for capsid assembly (Naumer M, et al., J Virol. 2012;86(23): 13038-13048. doi: 10.1128 / JVI.01675-12).While AAP is essential for nuclear localization of VP proteins and capsid assembly in AAV2, nuclear localization of AAP varies among 11 other serotypes and is dispensable for AAV4, AAV5, and AAV11 (Earley LF, et al. Adeno-associated virus (AAV) assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11. J Virol. 2017;91(3):1-21. doi:10.1128 / jvi.01980-16).

[0067] The Examples section below describes in detail compositions and methods for large-scale production of rAAV. In certain embodiments, a method for large-scale production of recombinant adeno-associated virus (rAAV) includes the steps of providing a vector encoding an AAV nucleic acid sequence or a closed, linear AAV nucleic acid sequence, culturing a human embryonic cell line in suspension, transfecting the vector encoding the AAV nucleic acid sequence and a transfection composition or the closed, linear AAV nucleic acid sequence and the transfection composition into the human cell line, incubating the transfected human cell line for approximately 50 to 100 hours, harvesting the transfected human cell line, and purifying the rAAV vector, thereby providing large-scale production of rAAV. In certain embodiments, the rAAV produced is a closed, linear rAAV.

[0068] Thus, in certain embodiments, a method for producing a high-titer population of recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences comprises transfecting 1×10 mammalian cells with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acid sequences encoding rep and cap genes, and (c) a heterologous transgene operably linked to at least one ITR and one or more regulatory elements, to produce a high-titer population of 1×10 rAAV cells. 6wherein the total amount of nucleic acid transfected from (a), (b), and (c) per cell is less than 1 μg; culturing the transfected cells for at least 40 hours; harvesting the transfected cells and purifying the rAAV vector particles produced, wherein the titer of rAAV is at least 9.3 x 10 13 Vector genomes / 3.0×10 9 transfected live cells.

[0069] AAV sequences can be obtained from various sources.For example, suitable AAV sequences can be obtained as described in WO 2005 / 033321 or from known sources, such as the American Type Culture Collection, or various academic vector core facilities.Alternatively, suitable sequences can be synthetically produced using known techniques with reference to published sequences.

[0070] AAV cap and rep sequences can be independently selected from different AAV parent sequences and introduced into host cells in a suitable manner known to those skilled in the art. In certain embodiments, rAAV particles have AAV capsid genes from AAV serotypes including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. In certain embodiments, rAAV particles have AAV capsid genes from AAV serotypes selected from the group consisting of AAV2, 3, 8, 9, and 10.

[0071] In certain embodiments, the rAAV particles comprise an AAV rep gene from an AAV serotype including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13. In certain embodiments, the rAAV particles have an AAV rep gene from an AAV serotype selected from the group consisting of AAV2, 3, 8, 9, and 10.

[0072] The present disclosure also provides a method for producing a population of purified recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences, the method comprising the steps of: transfecting a mammalian cell line suspended in culture medium with a transfection composition, the transfection composition comprising: (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acid sequences encoding rep and cap genes, and (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, and (d) a stable cationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is at least 1.5:1; culturing the transfected cell line for at least 40 hours; harvesting the transfected cell line of step (ii); and purifying the rAAV, wherein the purified virus is at least 2×10 4 It has a particle-to-infectivity ratio of less than vg / TCID50 and is devoid of prokaryotic DNA.

[0073] Mammalian cell lines used in embodiments of the present invention include suspension cells or cell lines, i.e., non-adherent cells or cell lines. In certain embodiments, the cell line is derived from a human fetal kidney cell line. In certain embodiments, the human fetal kidney cells lack SV40 antigens or other transforming antigens. In certain embodiments, the mammalian cell line is a suspension-adapted serum-free cell line. In certain embodiments, the cell line is derived from primary blood cells, such as lymphocytes, monocytes, macrophages, granulocytes, dendritic cells, and erythrocytes. In certain embodiments, the cell line is derived from a cell biopsy, including, for example, lymph node cells, bone marrow cells, and umbilical cord blood cells. In certain embodiments, the cell line is derived from circulating tumor cells. In certain embodiments, the cell line is derived from a blood cell line, such as Jurkat and Molt4 T cell lines, U937 and THP promonocytic cell lines, and B cell hybridomas. In certain embodiments, the cell line is derived from a stem cell.

[0074] Viral cell culture utilizes cells that contain, either stably or transiently, at least the minimal elements required to produce AAV particles, including the expression cassettes packaged into AAV capsids, AAV cap, and AAV rep or functional fragments thereof, and helper functions.

[0075] The cell also requires helper functions to package the AAV of the present invention. Optionally, these helper functions can be provided by herpesvirus. In another embodiment, the necessary helper functions are each provided by a human or non-human primate adenovirus source, available from a variety of sources, including, for example, the American Type Culture Collection (ATCC), Manassas, Va. (US). Various suitable adenovirus sequences have been reported. See, for example, chimpanzee adenoviruses C1 and C68 [U.S. Patent No. 6,083,716]; Pan 5, Pan 6, and Pan 7 [WO 02 / 33645], and hybrid adenoviruses, such as those reported in [e.g., WO 05 / 001103], and GenBank.

[0076] A variety of suitable cells and cell lines have been reported for use in AAV production. The cells themselves can be selected from any biological tissue, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Particularly desirable host cells are selected from any mammalian species, including, but not limited to, cells such as A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 cells (expressing functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2, Sf'c9, Sf-21, Tn368, BTI-Tn-5B1-4 (High-Five), and primary fibroblasts, hepatocytes, and myoblasts from mammals, including humans, monkeys, mice, rats, rabbits, and hamsters. The choice of mammalian species from which these cells are provided is not a limitation of the present invention, nor is the type of mammalian cell, ie, fibroblasts, hepatocytes, tumor cells, etc.

[0077] In a particular embodiment of any one of the aspects described herein, the host cell line is derived from the human embryonic kidney 293 cell line (HEK293).

[0078] The host cell may contain at least the minimal adenoviral DNA sequence required to express the E1A gene product, the E1B gene product, the E2A gene product, and / or the E4 ORF6 gene product. The host cell may contain other adenoviral genes, such as the VAI RNA, but these genes are not essential. The cell does not carry any adenoviral genes other than E1, E2A, and / or E4 ORF6, does not contain any other viral genes that could cause homologous recombination of contaminating viruses during the production of rAAV, and is capable of infection or transfection.

[0079] In a particular embodiment of any one of these aspects, the host cell lacks SV40 antigen or other transforming antigen. For example, when human embryonic kidney cells, such as HEK293 cells, are used as host cells, such cells may lack SV40 antigen or other transforming antigen.

[0080] Another type of host cell is one that is stably transformed with sequences encoding rep and cap and transfected with a construct carrying adenoviral E1, E2A, and E4 ORF6 DNA and the expression cassettes described above. Stable rep and / or cap-expressing cell lines, such as B-50 (International Patent Application Publication No. WO 99 / 15685) or those described in U.S. Pat. No. 5,658,785, can also be used. Another desirable host cell contains minimal adenoviral DNA sufficient to express E4 ORF6. Still other cell lines can be constructed using the novel modified cap of the present invention.

[0081] Preparation of the host cell employs techniques such as assembly of a selected DNA sequence. This assembly can be accomplished using conventional techniques. Such techniques are well known, including polymerase chain reaction, synthetic methods, and any other suitable method for providing the desired nucleotide sequence, including cDNA and genomic cloning as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0082] In certain embodiments, the host cell is a mammalian cell, i.e., the host cell line is a mammalian cell line.For example, the host cell, i.e., the host cell line is a human cell, for example, a human embryonic cell line.In any one particular embodiment of the aspects described herein, the host cell line is a human embryonic kidney cell line.

[0083] The cell culture work involved in rAAV production, including the expansion, seeding and transfection of adherent cells, is laborious and resource-consuming.Therefore, the use of cells suspended in aqueous liquid medium (" suspension cells ") for rAAV vector production is desirable in terms of its scalability and cost-effectiveness.Therefore, in any one particular embodiment of the aspects described herein, host cell line can be adapted to suspension.For example, host cell can be transfected with nucleic acid vector in suspension state.

[0084] Elements for AAV production (e.g., adenoviral E1a, E1b, E2a, and / or E4ORF6 gene products, rep or fragments thereof, cap, expression cassettes, and any other desired helper functions) can be delivered to packaging host cells individually or in combination in the form of any genetic element that transfers the sequences carried. As used herein, genetic element (vector) includes, for example, naked DNA, plasmids, phages, transposons, cosmids, episomes, proteins in nonviral delivery vehicles (e.g., lipid-based carriers), viruses, etc. that transfer the sequences carried. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. The methods used to construct any embodiment of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0085] One or more of the adenoviral genes can be stably integrated into the genome of the host cell or stably expressed as an episome. The promoter for each adenoviral gene can be independently selected from a constitutive promoter, an inducible promoter, or a native adenoviral promoter. The promoter can be regulated, for example, by the specific physiological state of the organism or cell (i.e., by its differentiation state or in replicating or resting cells) or by an externally added factor. Examples of such factors include, but are not limited to, antibiotics, cytokines, growth factors, hormones, etc.

[0086] In one embodiment, the stable or transient host cell contains the required elements under the control of an inducible or regulatable promoter, although the required elements may also be placed under the control of a constitutive or synthetic promoter.

[0087] Regulatory promoters allow gene expression to be controlled by externally supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions, such as acute phase, specific differentiation state of cells, or only in replicating cells.Regulatory promoters and systems are available from various commercial sources, including but not limited to Invitrogen, Clontech and Ariad.Many other systems have also been reported and can be easily selected by those skilled in the art. Examples of promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, and the T7 polymerase promoter system [WO 98 / 10088]; the ecdysone insect promoter [No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)]; the tetracycline-repressible system [Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)]; and the tetracycline-inducible system [Gossen et al., Science, 268:1766-1769 (1995); Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)]. (1998)], the RU486-inducible system [Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)], and the rapamycin-inducible system [Magari et al., J Clin. Invest., 100:2865-2872 (1997)]. Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0088] In certain instances, native promoters are used.Native promoters can be used when it is desired that the expression of gene products mimic native expression.Native promoters can be used when the expression of desired transgenes must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli.Other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

[0089] In the case where transgene is included, transgene is functionally linked to tissue-specific promoter.For example, if expression is desired in skeletal muscle, promoters that are active in muscle should be used.These include, but are not limited to, promoters derived from the gene encoding skeletal β-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, and synthetic muscle promoters that have higher activity than naturally occurring promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters that are tissue-specific include, inter alia, liver (albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), lymphocyte (CD2, Hansal et al., J. Immunol., 161:1063-8 (1996)), and IL-16 (IL-16). (1998); immunoglobulin heavy chain; T cell receptor alpha chain), neuronal, e.g., neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and neuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)). With regard to liver-specific promoters, examples include HLP, LP1, HCR-hAAT, ApoE-hAAT, and LSP.These promoters are described in more detail in the following references: HLP: McIntosh J. et al., Blood 2013 Apr. 25, 121(17):3335-44; LP1: Nathwani et al., Blood. 2006 April 1, 107(7):2653-2661; HCR-hAAT: Miao et al., Mol Ther. 2000;1:522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-645(1996); and LSP: Wang et al., Proc Natl Acad Sci USA. 1999 March 30,96(7):3906-3910. See also Brown HC et al., Mol. Ther.: Meth. Clin. Dev. Vol. 9, pp:57-91, June 2018.

[0090] Examples of suitable activatable and constitutive promoters are known to those skilled in the art. In yet another alternative, the selected stable host cell can contain selected elements under the control of a constitutive promoter and other selected elements under the control of one or more inducible promoters. For example, stable host cells can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells can be generated by those skilled in the art.

[0091] Closed-ended linear double-stranded nucleic acid A closed linear DNA molecule typically contains covalently closed ends, also referred to as hairpin loops, where there is no base pairing between complementary DNA strands. This hairpin loop connects the ends of the complementary DNA strands. This type of structure typically forms at the telomeric ends of chromosomes to protect chromosomal DNA from loss or damage by trapping the terminal nucleotides within the closed structure. In the example of a closed linear DNA molecule described herein, the hairpin loop is adjacent to a complementary base-paired DNA strand, forming a closed linear (cl) DNA-shaped structure. A closed linear DNA molecule includes barbell-shaped DNA.

[0092] One or more of nucleic acids (a)-(c), i.e., (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acid sequences encoding the rep and cap genes, and (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, can be present on a closed-ended, linear, double-stranded nucleic acid. Such nucleic acids can be produced by a variety of known methods, including in vitro cell-free synthesis and in vivo methods.

[0093] In certain embodiments, the nucleic acid sequence comprising one or more of (a), (b), or (c) is an amplified linear open-end DNA with blunt ends or overhangs, and a synthesized hairpin molecule is ligated to one or both ends to form a closed-end linear DNA comprising one or more of the nucleic acids of (a), (b), or (c). The unligated hairpin is purified using means well known to those skilled in the art. The DNA is amplified by PCR and ligated into a double-stranded form.

[0094] One method for generating covalently closed-ended linear double-stranded nucleic acids is by incorporating protelomerase binding sites into a precursor molecule such that the protelomerase binding sites are adjacent to the nucleic acid of interest. The nucleic acid of interest can contain one or more of (a), (b), and (c), i.e., (a), (b), and (c); any combination of (a), (b), and (c); or only (a), only (b), or only (c); exposure of the molecule to protelomerase cleaves and ligates the DNA at the sites. Non-limiting examples of cell-free in vitro synthesis are described, for example, in US 9,109,250; US 6,451,563; Nucleic Acids Res. 2015 Oct 15; 43(18): el20; US 9499847; 15 / 508,766; PCT / GB2017 / 052413; and Antisense & nucleic acid drug development 11:149-153 (2001), which are incorporated by reference in their entireties. DNA from cell-free in vitro synthesis does not have any prokaryotic DNA modifications.

[0095] The recombinant AAV vector genome can be designed to have at least one of wild-type ITRs, synthetic ITRs, or DD ITRs, or a combination thereof, flanked by an imperfect palindrome containing a protelomerase site, e.g., telRL. A template is used to generate a closed, linear, double-stranded nucleic acid vector when cleaved by telomerase to form covalently closed ends. In one embodiment, the vector contains two DD ITRs, an expression cassette, and telomerase binding sites flanking each side of the DD ITRs, which can be cleaved by telomerase to form covalently closed ends. The closed linear DNA contains half of the protelomerase binding site.

[0096] Additionally, prokaryotic systems can be used. In lysogens, bacteriophage N15 exists as a linear extrachromosomal DNA with covalently closed ends (see Rybchin VN, Svarchevsky AN (1999) The plasmid prophage N15: a linear DNA with covalently closed ends. Mol Microbiol 33:895-903). This DNA is generated by a cleavage-joining reaction performed by a single enzyme, protelomerase, e.g., TelN (prokaryotic telomerase) [Deneke J, Ziegelin G, Lurz R, Lanka E (2000) The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity. Proc Natl Acad Sci USA 97:7721-7726]. Protelomerases, e.g., TelN, recognize target sequences within double-stranded DNA. The target site is an imperfect palindrome called telRL, formed by two halves, telR and telL, that correspond to the covalently closed ends of a linear prophage. This enzyme cleaves both DNA strands and joins the resulting ends to form a covalently closed hairpin structure. The resulting DNA molecule contains two hairpin loops. TelN can linearize recombinant plasmids containing telRL sites [Deneke J, et al., (2000). Proc Natl Acad Sci USA 97:7721-7726]. Therefore, this enzyme can be used in plasmid DNA for expression in higher organisms.

[0097] In certain embodiments, in vivo cell system is used to produce closed-ended linear double-stranded nucleic acid.This method includes using cells that express protelomerase, such as TelN or other protelomerase, and the protelomerase gene is placed under the control of a regulable promoter.For example, an inducible promoter, such as a small molecule regulable promoter or a temperature-sensitive promoter, such as a heat shock promoter.After sufficient production of AAV template DNA, or other nucleic acid of interest, or a combination thereof, protelomerase can be expressed, which will excise the nucleic acid of interest from the template, for example, the nucleic acid comprising one or more of (a) helper, (b) rep / cap, or (c) AAV genome.

[0098] In a specific embodiment, an in vivo cell system is used to produce a non-viral DNA vector construct for delivering a predetermined nucleic acid sequence to a target cell for sustained expression. The non-viral DNA vector comprises two DD-ITRs, each of which comprises an inverted terminal repeat (DTR) having regions A, A', B, B', C, C', and D; a D' region, in which the D and D' regions are complementary palindromic sequences approximately 5 to 20 nt in length and are adjacent to the A and A' regions; and a predetermined nucleic acid sequence (e.g., a heterologous gene to be expressed), in which the two DD-ITRs flank the nucleic acid in a covalently closed non-viral DNA, and the closed linear vector contains one and two protelomerase binding sites at each end.

[0099] The TelN / telRL system described herein can be used to generate closed, linear DNA fragments either by linearizing a parental plasmid containing a single telRL site, or by excising an rAAV DNA fragment or non-viral vector fragment containing a promoter, gene of interest, and polyadenylation signal from a parental plasmid with two flanking ITRs and further containing two telRL sites flanking each segment. In one embodiment, at least one double "D" ITR is present. The resulting linear, covalently closed DNA molecule is functional in vivo.

[0100] The system comprises a recombinant host cell. Host cells suitable for use in the present production system include microbial cells, such as bacterial cells, for example, E. coli cells, and yeast cells, for example, S. cerevisiae. Mammalian host cells may also be used, including Chinese hamster ovary (CHO) cells of the K1 line (ATCC CCL 61), including, for example, the Pro5 variant (ATCC CRL 1281); fibroblast-like cells derived from SV40-transformed African green monkey kidney of the CV-1 line (ATCC CCL 70), COS-1 line (ATCC CRL 1650), and COS-7 line (ATCC CRL 1651); human cancer cells, including mouse L cells, mouse 3T3 cells (ATCC CRL 1658), mouse C127 cells, human embryonic kidney cells of the 293 line (ATCC CRL 1573), those of the HeLa line (ATCC CCL 2), and neuroblastoma cells of the IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10), and SK-N-SH (ATCC HTB 11) strains.

[0101] The host cell is designed to encode at least one recombinase. The host cell may also be designed to encode two or multiple recombinases. The term "recombinase" refers to an enzyme that catalyzes DNA exchange at a specific target site, e.g., a palindromic sequence, by excision / insertion, inversion, translocation, and exchange. Examples of recombinases suitable for use in this system include, but are not limited to, TelN, Tel, Tel (gp26 K02 phage), Cre, Flp, phiC31, Int, and other lambda phage integrases, e.g., phi80, HK022, and HP1 recombinases. The target sequences for each of these recombinases are respectively as follows: telRL part: TIFF2026032042000001.tif11143; pal site: TIFF2026032042000002.tif4143; φK02 telRL part: TIFF2026032042000003.tif4128; loxP site: TIFF2026032042000004.tif4128; FRT part: TIFF2026032042000005.tif4128; phiC31 attP site: TIFF2026032042000006.tif18145; and λattP site: TIFF2026032042000007.tif18145.

[0102] Expression of the recombinase is under the control of any regulated or inducible promoter, i.e., a promoter that is activated under specific physical or chemical conditions or stimuli. Examples of suitable promoters include thermoregulated promoters, such as the λpL promoter, the IPTG-regulated lac promoter, the glucose-regulated ara promoter, the T7 polymerase-regulated promoter, the cold-shock-inducible cspA promoter, a pH-inducible promoter, or combinations thereof, such as the tac (T7 and lac) dual-regulated promoter.

[0103] Other methods for producing covalently closed linear DNA lacking bacterial sequences are known in the art, such as by forming minicircular DNA from a plasmid (e.g., as described in U.S. Pat. Nos. 8,828,726 and 7,897,380, the entire contents of each of which are incorporated by reference). For example, one cell-free synthesis method combines the use of two enzymes, Phi29 DNA polymerase and protelomerase, to produce a high-fidelity, covalently closed linear DNA construct. This construct does not contain antibiotic resistance markers, thus eliminating the packaging of these sequences. This process can amplify AAV genomic DNA on a commercial scale in a two-week process and maintain the ITR sequences required for virus production.

[0104] Phi29 DNA polymerase is used to amplify double-stranded DNA by rolling circle amplification, and protelomerase is used to generate covalently closed linear DNA, which, combined with a state-of-the-art purification process, produces a pure DNA product containing only the sequence of interest. Phi29 DNA polymerase has high fidelity (1 × 10 6 ~1×10 7) and high processivity (approximately 70 kbp). These characteristics make this polymerase particularly suitable for large-scale production of GMP DNA. Protelomerase (also known as telomere resolvase) catalyzes the formation of covalently closed hairpin ends on linear DNA and has been identified in several phages, bacterial plasmids, and bacterial chromosomes. Paired protelomerases recognize inverted palindromic DNA recognition sequences and catalyze strand cleavage, strand exchange, and DNA ligation to generate closed, linear hairpin ends. The formation of these closed-ended structures renders the DNA resistant to exonuclease activity, thereby allowing for easier purification and potentially improving stability and expression duration.

[0105] Protelomerase binding site In one embodiment, the DNA construct comprises a protelomerase binding site, and the covalently closed ends are formed by protelomerase enzymatic activity (e.g., in vitro). Protelomerase binding sites and corresponding protelomerases used in the present invention are provided in U.S. Patent No. 9,499,847, the entire contents of which are incorporated herein by reference. The protelomerase target sequence used in the present invention preferably comprises a double-stranded palindromic (perfect inverted repeat) sequence at least 14 base pairs in length. Preferred perfect inverted repeat sequences include those of SEQ ID NOs: 1-6 and variants thereof. TIFF2026032042000008.tif4128 is a 22-base consensus sequence for perfect inverted repeats of mesophilic bacteriophages. The base pairs of perfect inverted repeats are conserved among different bacteriophages at certain positions, and sequence flexibility may be found at other positions. Thus, SEQ ID NO: 1 is the minimal consensus sequence for perfect inverted repeat sequences to be used with bacteriophage protelomerase in the process of the present invention.

[0106] Within the consensus defined by SEQ ID NO:1, TIFF2026032042000009.tif4128 is a perfect inverted repeat sequence used with Escherichia coli phage N15 and Klebsiella phage Phi KO2 protelomerases. Also included are SEQ ID NOs:3-5: within the consensus defined by SEQ ID NO:1. TIFF2026032042000010.tif25145 are particularly preferred perfect inverted repeat sequences for use with protelomerases from Yersinia phage PY54, Halomonas phage phiHAP-1, and Vibrio phage VP882, respectively. TIFF2026032042000011.tif4128 is a perfect inverted repeat sequence particularly preferred for use with Borrelia burgdorferi protelomerase. This perfect inverted repeat sequence is derived from lpB31.16, a linear, covalently closed plasmid contained in Borrelia burgdorferi. This 14-base sequence is shorter than the 22-bp consensus perfect inverted repeat of bacteriophages (SEQ ID NO:1), indicating that bacterial protelomerases may differ from bacteriophage protelomerases in their requirements for specific target sequences. However, all protelomerase target sequences share the common structural motif of a perfect inverted repeat.

[0107] The perfect inverted repeat sequence may be greater than 22 bp in length, depending on the requirements of the particular protelomerase used in the process of the invention. Thus, in some embodiments, the perfect inverted repeat may be at least 30, at least 40, at least 60, at least 80, or at least 100 base pairs in length. Examples of such perfect inverted repeat sequences include SEQ ID NOs: 7-9 and variants thereof. TIFF2026032042000012.tif32146SEQ ID NOs:7-9 and variants thereof are particularly preferred for use with protelomerases from Vibrio phage VP882, Yersinia phage PY54, and Halomonas phage phi HAP-1, respectively.

[0108] The perfect inverted repeat may be flanked by additional inverted repeat sequences. The flanking inverted repeats may be perfect or imperfect repeats, i.e., perfectly symmetric or partially symmetric. The flanking inverted repeats may be contiguous or non-contiguous with respect to the central palindrome. The protelomerase target sequence may comprise an imperfect inverted repeat sequence comprising a perfect inverted repeat sequence at least 14 base pairs in length. An example is SEQ ID NO: 14. The imperfect inverted repeat sequence may comprise a perfect inverted repeat sequence at least 22 base pairs in length. An example is SEQ ID NO: 10.

[0109] In certain embodiments, the protelomerase target sequence comprises the sequence of SEQ ID NOs: 10-14 or a variant thereof. TIFF2026032042000013.tif59146

[0110] The sequences of SEQ ID NOs:10-14 contain perfect inverted repeats as defined above and further contain flanking sequences from related organisms. A protelomerase target sequence containing SEQ ID NO:10 or a variant thereof is preferred for use in combination with Escherichia coli N15 TelN protelomerase and variants thereof. A protelomerase target sequence containing SEQ ID NO:11 or a variant thereof is preferred for use in combination with Klebsiella phage Phi K02 protelomerase and variants thereof. A protelomerase target sequence containing SEQ ID NO:12 or a variant thereof is preferred for use in combination with Yersinia phage PY54 protelomerase and variants thereof. A protelomerase target sequence containing SEQ ID NO:13 or a variant thereof is preferred for use in combination with Vibrio phage VP882 protelomerase and variants thereof. A protelomerase target sequence containing SEQ ID NO:14 or a variant thereof is preferred for use in combination with Borrelia burgdorferi protelomerase.

[0111] Variants of any of the above palindromic sequences or protelomerase target sequences include homologs or mutants thereof. Variants include truncations, substitutions, or deletions of the native sequence. A variant sequence is any sequence whose presence in a DNA template allows its conversion to closed, linear DNA by the enzymatic activity of protelomerase. This can be easily determined by using an appropriate assay for the formation of closed, linear DNA. Any appropriate assay reported in the art can be used. An example of a suitable assay is described in Deneke et al., PNAS (2000) 97, 7721-7726. In certain embodiments, variants achieve protelomerase binding and activity comparable to that observed with the native sequence. Examples of preferred variants of the palindromic sequences described herein include truncated palindromic sequences that retain the perfect repeat structure and are still capable of forming closed, linear DNA. However, variant protelomerase target sequences can be modified so that they no longer retain the perfect palindrome, as long as they can act as substrates for protelomerase activity.

[0112] It should be understood that one of skill in the art can readily identify suitable protelomerase target sequences for use in the present invention based on the structural principles outlined above. Candidate protelomerase target sequences can be screened for their ability to promote the formation of closed linear DNA using the assays described above.

[0113] Generation of covalently closed linear DNA constructs The covalently closed vector described herein can be produced in vitro or in vivo. The vector is a covalently closed linear double-stranded vector that can express a transgene in target cells. For example, one example of an in vitro process for producing a closed linear expression cassette DNA, including the ITRs described herein, includes: (a) contacting a DNA template containing at least one expression cassette flanked on either side by a protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions that promote the amplification of the template; and (b) contacting the amplified DNA produced in (a) with at least one protelomerase under conditions that promote the formation of a closed linear expression cassette DNA. The closed linear expression cassette DNA product can comprise, consist of, or essentially consist of a eukaryotic promoter operably linked to the coding sequence of interest, and optionally, a eukaryotic transcription termination sequence. The closed linear expression cassette DNA product may further lack one or more bacterial or vector sequences, typically selected from the group consisting of: (i) a bacterial origin of replication, (ii) a bacterial selectable marker (typically an antibiotic resistance gene), and (iii) an unmethylated CpG motif.

[0114] As outlined above, any DNA template containing at least one protelomerase target sequence can be amplified according to the process of the present invention. Thus, while the production of therapeutic DNA molecules, e.g., for DNA vaccines or other therapeutic proteins and nucleic acids, is preferred, the process of the present invention can be used to produce any type of closed, linear DNA. The DNA template can be double-stranded (ds) or single-stranded (ss) DNA. The double-stranded DNA template can be open, circular, closed, linear, or closed. Preferably, the template is closed, circular, double-stranded DNA. Closed, circular dsDNA templates are particularly preferred for use with RCA (rolling circle amplification) DNA polymerases. Circular dsDNA templates can be in the form of plasmids or other vectors, typically used to house genes for bacterial propagation. Thus, the process of the present invention can be used to amplify any commercially available plasmid or other vector, e.g., a commercially available DNA drug, and then convert the amplified vector DNA into closed, linear DNA.

[0115] Open circular dsDNA can be used as a template when the DNA polymerase is a strand-displacing polymerase capable of initiating amplification from a nicked DNA strand. In this embodiment, the template can be pre-incubated with one or more enzymes that nick one or more sites in the template DNA strand. Closed linear dsDNA can also be used as a template. The closed linear dsDNA template (starting material) can be identical to the closed linear DNA product. When closed linear DNA is used as a template, it can be incubated under denaturing conditions to form single-stranded circular DNA before or during conditions that promote amplification of the template DNA. In one embodiment, closed-ended linear double-stranded DNA is produced in eukaryotic cells, such as insect cells described in PCT publications WO 2019032102 and WO 2019169233. In one embodiment, the DNA is not produced in eukaryotic cells, and the DNA lacks eukaryotic sequences. In one embodiment, the closed-ended linear double-stranded DNA vector is produced as described in PCT Publication WO 2019143885.

[0116] As outlined above, the DNA template typically comprises an expression cassette as described above, consisting of or consisting essentially of a eukaryotic promoter operably linked to a sequence encoding a protein of interest and optionally a eukaryotic transcription termination sequence. Optionally, the expression cassette may be a minimal expression cassette as defined above, that is, lacking one or more bacterial or vector sequences, typically selected from the group consisting of (i) a bacterial replication origin, (ii) a bacterial selection marker (typically an antibiotic resistance gene), and (iii) an unmethylated CpG motif.

[0117] Cell culture medium As used herein, the terms "cell culture medium" and "culture medium" refer to a nutrient solution used to grow cells in vitro, typically providing at least one component from one or more of the following categories: 1) an energy source, usually in the form of carbohydrates, e.g., glucose; 2) one or more of all essential amino acids, usually a basis set of 20 amino acids; 3) vitamins and / or other organic compounds required at low concentrations; 4) free fatty acids; and 5) trace elements, typically defined as inorganic compounds or naturally occurring elements required at very low concentrations, usually in the micromolar range. The nutrient solution can optionally be supplemented with additional components to optimize cell growth and / or transfection.

[0118] The cell cultures of the present invention are prepared in a medium suitable for the particular host cells being cultured. Appropriate cell culture media that can be used to culture a particular cell type will be apparent to those skilled in the art. Exemplary commercially available media include, for example, Ham's F10 (SIGMA), Minimum Essential Medium (MEM, SIGMA), RPMI-1640 (SIGMA), Dulbecco's Modified Eagle's Medium (DMEM, SIGMA); Iscove's Modified Dulbecco's Medium (Gibco) containing 10% fetal bovine serum (see Xiao et al., "Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus," J. Virol., 72: 2224-2232 (1998)), and DMEM / F12 (Life Technologies). Any of these or other suitable media may be supplemented, as needed, with hormones and / or other growth factors (e.g., without limitation, insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics (e.g., puromycin, neomycin, hygromycin, blasticidin, or gentamicin™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), lipids (e.g., linoleic acid or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source, and / or may be modified, as described herein, to promote the production of recombinant glycoproteins having a low mannose content.

[0119] Depending on the requirements of the particular cell line or method used, cell culture media may contain serum supplements, such as fetal bovine serum, or serum substitutes. Examples of serum substitutes (for serum-free growth of cells) are TCH™, TM-235™, and TCH™, which are commercially available from Celox (St. Paul, Minn.) and KOSR (Knockout (KO) Serum Replacer; Life Technologies).

[0120] In a particular embodiment of any one of these aspects, the host cells can be grown in serum-free, protein-free, growth factor-free, and / or peptone-free medium. The term "serum-free" as applied to a medium generally includes any mammalian cell culture medium that does not contain serum, such as fetal bovine serum (FBS). The term "growth factor-free" as applied to a medium includes any medium to which exogenous growth factors (e.g., insulin, IGF-1) are not added. The term "peptone-free" as applied to a medium includes any medium to which exogenous protein hydrolysates, such as animal and / or plant protein hydrolysates, are not added.

[0121] In any one particular embodiment of these aspects, the cell culture medium is serum-free. By "serum-free" it is understood that the serum concentration in the medium is preferably less than 0.1% (v / v), more preferably less than 0.01% (v / v). By "essentially serum-free" it is meant that less than about 2% (v / v) serum is present, more preferably less than about 1% serum is present, even more preferably less than about 0.5% (v / v), and even more preferably less than about 0.1% (v / v). When a serum-free defined medium is used, the medium is usually enriched with certain amino acids, vitamins and / or trace elements (see, for example, U.S. Patent No. 5,122,469 to Mather et al. and U.S. Patent No. 5,633,162 to Keen et al.).

[0122] "Culturing" or "incubating" (as used interchangeably with respect to growing, transforming, and / or maintaining a host cell or host cell line) is conducted under conditions of sterility, temperature, pH, atmospheric gas components (e.g., oxygen, carbon dioxide, dinitrogen), humidity, culture vessel, culture volume, passaging, operation, and other parameters suitable for the intended purpose and conventionally known in the art of mammalian cell culture.

[0123] In certain embodiments, the culture medium contains an amino acid at a concentration of about 1 mM to about 100 mM. For example, the culture medium contains an amino acid at a concentration of about 1 mM to about 20 mM, e.g., about 5 mM to about 15 mM. In certain embodiments, the culture medium contains an amino acid at a concentration of about 7.5 mM to about 12.5 mM. For example, the culture medium contains an amino acid at a concentration of about 10 mM.

[0124] In certain embodiments, the culture medium comprises L-glutamine or an L-glutamine-containing dipeptide. An exemplary L-glutamine-containing dipeptide is L-alanyl-L-glutamine (e.g., GLUTAMAX™). Typically, the culture medium comprises L-glutamine or an L-glutamine-containing dipeptide at a concentration of about 1 mM to about 100 mM. For example, the culture medium comprises L-glutamine or an L-glutamine-containing dipeptide at a concentration of about 1 mM to about 20 mM, e.g., about 5 mM to about 15 mM. In certain embodiments, the culture medium comprises L-glutamine or an L-glutamine-containing dipeptide at a concentration of about 7.5 mM to about 12.5 mM. For example, the culture medium comprises L-glutamine or an L-glutamine-containing dipeptide at a concentration of about 10 mM.

[0125] In certain embodiments, the culture medium can also contain nonionic surfactant polyol or surfactant.Exemplary nonionic surfactants include but are not limited to polysorbates, such as polysorbate 20 (TWEEN 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 188, poloxamer 407; polyethylene polypropylene glycol; or polyethylene glycol (PEG).In some embodiments of any one of these aspects, the nonionic surfactant polyol or surfactant is poloxamer. Exemplary poloxamers are Poloxamer 188 (P188), Pluronic® F127, Pluronic® F38, Pluronic® F68, Pluronic® F87, Pluronic® F108, Pluronic® 10R5, Pluronic® 17R2, Pluronic® 17R4, Pluronic® 25R2, Pluronic® 25R4, Pluronic® 31R1, Pluronic® F108 Cast Solid Surfactant, Pluronic® F108 NF, Pluronic® F108 Pastille, Pluronic® F108NF Prill, Poloxamer 338, Pluronic® F127 NF, Pluronic® F127 NF 500 BHT Prills, Pluronic® F127 NF Prills Poloxamer 407, Pluronic® F38 Pastilles, Pluronic® F68 LF Pastilles, Pluronic® F68 NF, Pluronic® F68 NF Prills, Pluronic® F68 Pastilles, Pluronic® F77, Pluronic® F77 Micropastiles, Pluronic® F87 NF, Pluronic® F87 NF Prills Poloxamer 237, Pluronic® F 88, Pluronic® F88 Pastilles, Pluronic® F98, Pluronic® FT L 61, Pluronic® L10, Pluronic® L101, Pluronic® L121, Pluronic® L31, Pluronic® L35, Pluronic® L43, Pluronic® L61, Pluronic® L62, Pluronic® L62 LF, Pluronic® L62D, Pluronic® L64, Pluronic® L81, Pluronic® L92, Pluronic® L44 NF INH surfactants include, but are not limited to, poloxamer 124, Pluronic® N3, Pluronic® P103, Pluronic® P104, Pluronic® P105, Pluronic® P123 surfactant, Pluronic® P65, Pluronic® P84, Pluronic® P85, and the like.

[0126] The amount of nonionic surfactant polyol or surfactant in the culture medium can be at least about 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05% (w / w, w / v, or v / v) or more. For example, the amount of nonionic surfactant polyol or surfactant in the culture medium can range from about 0.001% to about 1% (weight / volume). For example, the culture medium contains a nonionic surfactant polyol or surfactant at a concentration of about 0.01% to about 0.5%, about 0.015% to about 0.45%, about 0.02% to about 0.4%, or about 0.025% to about 0.35%. 0.1%. For example, the culture medium contains a non-ionic surfactant polyol or detergent at a concentration of about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, or about 0.05%.

[0127] In certain embodiments, the culture medium contains an antifoaming agent. The term "antifoaming agent" refers to a chemical substance that, when added to a liquid, can substantially reduce the surface activity of the liquid, thereby substantially preventing the liquid from foaming. Exemplary antifoaming agents that can be used in the present invention include, but are not limited to, high molecular weight silicones and other materials known in the art for such applications.

[0128] The amount of antifoam agent in the culture medium can be at least about 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05% (w / w, w / v, or v / v) or more. For example, the amount of antifoam agent in the culture medium can range from about 0.001% to about 1% (weight / volume). For example, the culture medium contains an antifoam agent at a concentration of about 0.01% to about 0.5%, about 0.015% to about 0.45%, about 0.02% to about 0.4%, or about 0.025% to about 0.35%. 0.1%. For example, the culture medium comprises an antifoaming agent at a concentration of about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, or about 0.05%.

[0129] As used herein, the term "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. In many cases, a cell line is a clonal population derived from a single ancestral cell. Furthermore, it is known in the art that spontaneous or induced changes in karyotype may occur during the storage or transfer of such a clonal population. Thus, cells derived from a referenced cell line may not be strictly identical to their ancestral cells or cultures, and the referenced cell line includes such variants.

[0130] Host cell lysis In any one particular embodiment of these aspects, this method comprises lysing transfected host cells.Methods for lysing host cells in cell culture are well known in the art.For example, non-ionic surfactant can be added to cell culture or cell culture supernatant.Usually, non-ionic surfactant is added to cell culture to a final concentration of at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1% (w / v, w / w or v / v) or more. For example, nonionic surfactant is added to a cell culture to a final concentration of about 0.05% to about 1%, about 0.1% to about 0.95%, about 0.15% to about 0.9%, about 0.2% to about 0.85%, about 0.25% to about 0.8%, about 0.3% to about 0.75%, about 0.35% to about 0.65%, about 0.4% to about 0.6%, or 0.45% to about 0.55%. In some embodiments, non-ionic surfactant is added to cell culture to a final concentration of about 0.05%, 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95% or about 1%.For example, non-ionic surfactant can be added to cell culture to a final concentration of about 0.5%.

[0131] Usually, the non-ionic surfactant is mixed with the cell culture for a period sufficient to dissolve the host cells present in the cell culture or cell culture supernatant.For example, the non-ionic surfactant is mixed with the cell culture for a period of about 15 minutes to about 2 hours.In some embodiments, the non-ionic surfactant is mixed with the cell culture for a period of about 30 minutes to about 60 minutes.

[0132] Mixing can be performed at ambient temperature or elevated temperature. For example, mixing with a non-ionic surfactant can be performed at a temperature of about 15°C to about 37°C. In some embodiments, mixing with a non-ionic surfactant can be performed at a temperature of about 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 28°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.

[0133] It should be noted that any desired non-ionic surfactant can be used to lyse transfected host cells. Exemplary non-ionic surfactants and classes of non-ionic surfactants for lysing transfected host cells include polyallylphenol polyethoxy ethers; polyalkylphenol polyethoxy ethers; polyglycol ether derivatives of saturated fatty acids; polyglycol ether derivatives of unsaturated fatty acids; polyglycol ether derivatives of fatty alcohols; polyglycol ether derivatives of alicyclic alcohols; fatty acid esters of polyoxyethylene sorbitan; alkoxylated vegetable oils; alkoxylated acetylenic diols; polyalkoxylated alkylphenols; fatty acid alkoxylates; sorbitan alkoxylates; sorbitol esters; C8-C9 22The non-ionic surfactant for lysing host cells may comprise alkyl or alkenyl polyglycosides; polyalkoxystyryl ethers; alkylamine oxides; block copolymer ethers; polyalkoxylated fatty acid glycerides; polyalkylene glycol ethers; linear aliphatic or aromatic polyesters; organic silicones; polyallylphenols; sorbitan ester alkoxylates; and ethylene glycol mono- and diesters and their mixtures; ethoxylated tristyrylphenols; ethoxylated fatty alcohols; ethoxylated lauryl alcohols; ethoxylated castor oils; and ethoxylated monylphenols; alkoxylated alcohols, amines or acids. In some embodiments of any one of these aspects, the non-ionic surfactant for lysing host cells may be selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof.

[0134] Certain exemplary non-ionic surfactants for lysing transfected host cells include ECOSURF EH-9, polysorbates (e.g., polysorbate 20 (TWEEN 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85), ECOSURF EH-14, TWEEN 60 non-ionic surfactant, PPG-PEG-PPG Pluronic 10R5, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, Brij S20, Brij S10, Brij O10, Brij C10, BRIJ The non-ionic surfactants include, but are not limited to, O20, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, and TERGITOL NP-13, and any combination thereof. In some embodiments, the non-ionic surfactant for lysing transfected host cells is not Triton X-100.

[0135] In some embodiments, amphoteric surfactants can be added to cell cultures to lyse transfected host cells. Exemplary amphoteric surfactants include sulfonates, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO (3-{(3-cholamidopropyl)dimethylammonio}-2-hydroxy-1-propanesulfonate), 3-(decyldimethylammonio)propanesulfonate, 3-(dodecyldimethylammonio)propanesulfonate, 3-(N,N-dimethylmyristylammonium ... ammonium phosphates, such as lecithin; ammonium phosphates, such as ammonium phosphates, ...

[0136] In some embodiments, surfactant, for example, amphoteric surfactant, can be amine oxide surfactant.For example, amine oxide surfactant can be added to cell culture to lyse host cells.The amine oxide surfactant that can be used in the method described herein is trialkylamine N-oxide, for example, 1 R 2 R 3 and the amine oxide of NO, where R 1 is a substituted or unsubstituted alkyl or alkenyl containing from about 8 to about 30 carbon atoms, and R 2 and R 3 are independently substituted or unsubstituted alkyl or alkenyl groups containing from about 1 to about 18 carbon atoms. Non-limiting examples of trialkylamine N-oxides and trialkylamine N-oxide surfactants that may be used are described in WO1998055581, which is incorporated herein by reference in its entirety.

[0137] The lysate may contain impurities, such as host cell DNA (hcDNA). Therefore, this method may include a post-lysis step to remove or reduce the amount of impurities, such as hcDNA, from the lysate before isolating / purifying rAAV. Methods and compositions for reducing the amount of host cell DNA in cell culture or cell culture supernatant are well known in the art. For example, cationic amines or nucleases can be added to the lysate.

[0138] In some embodiments, the post-lysis step includes adding a selective sedimentation agent to reduce or remove impurities, such as hcDNA, from the lysate. As used herein, "selective sedimentation agent" refers to any agent, compound, etc. that, when added to a preparation containing a population of recombinant viral particles and contaminating nucleic acid molecules, results in the selective precipitation of at least a substantial amount of the contaminating nucleic acid molecules from the recombinant viral particles. Exemplary agents added to the lysate in the post-lysis step include, but are not limited to, cetyltriethylammonium bromide, cetylpyridinium chloride, benzethonium chloride, tetradecyltrimethylammonium chloride, polyethyleneimine, and combinations thereof.

[0139] In some embodiments, nucleases, such as endonucleases, are added to the lysate to reduce or remove impurities, such as hcDNA. Exemplary endonucleases include endonucleases from both prokaryotes and eukaryotes. In some embodiments, the nuclease is BENZONASE® or salt-active nuclease (SAN).

[0140] Typically, the nuclease is added to the lysate to a final concentration of at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1% (w / v, w / w or v / v) or more. For example, nuclease is added to the lysate to a final concentration of about 0.05% to about 1%, about 0.1% to about 0.95%, about 0.15% to about 0.9%, about 0.2% to about 0.85%, about 0.25% to about 0.8%, about 0.3% to about 0.75%, about 0.35% to about 0.65%, about 0.4% to about 0.6%, 0.45% to about 0.55%, about 0.05% to about 0.4%, or about 0.2% to about 0.4%. In some embodiments, the nuclease is added to the lysate to a final concentration of about 0.05%, 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1%. For example, the nuclease is added to the lysate to a final concentration of about 0.2%. In some embodiments, the nuclease may be added to the lysate to a final concentration of about 0.05% to about 0.4%.

[0141] Typically, the agent or nuclease is mixed with the lysate for about 15, 20, 30, 35, 40, 45, 50, 55 minutes or longer. In some embodiments, the agent or nuclease is mixed with the lysate for about 10 minutes to about 4 hours. For example, the agent or nuclease is mixed with the lysate for about 15 minutes to about 3 hours. In some embodiments, the agent or nuclease is mixed with the lysate for about 30 minutes to about 120 minutes. For example, the agent or nuclease is mixed with the lysate for about 30 minutes.

[0142] In some embodiments, the method includes clarifying the lysate, for example, the method includes clarifying the lysate by depth filtration to produce a clarified composition.

[0143] rAAV isolation / purification A variety of methods for isolating / purifying rAAV from the lysate derived from host cell line are known in the art.Such methods include but are not limited to density gradient, tangential flow filtration, affinity chromatography, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydroxylapatite chromatography, hydrophobic interaction chromatography, and various combinations thereof.Exemplary methods for isolating / purifying rAAV from host cell lysate are described in, for example, U.S. Patent No. 6,592,123; U.S. Patent No. 9,862,936; International Patent Publication No. WO2019 / 241535; International Patent Publication No. WO2005 / 035743; and International Patent Publication No. WO2019 / 212921, the entire contents of which are incorporated herein by reference.

[0144] Aspects of the present invention can be represented by the following numbered embodiments 1-103. Embodiment 1: A method for producing a recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences, comprising: (i) optionally culturing a human embryonic cell line in suspension; (ii) transfecting a human embryonic cell line with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising: (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication; (b) a nucleic acid sequence encoding the AAV rep and AAV cap genes; and (c) at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements; (iii) incubating the transfected human cell line for about 40 to 400 hours; and (iv) lysing the transfected human cell line and purifying the nucleic acid sequence encoding the rAAV. thereby producing rAAV. Embodiment 2: The method of claim 1, wherein the cells of said cell line are transfected in suspension. Aspect 3: The method according to any one of claims 1 to 2, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human fetal kidney cell line. Embodiment 4: The method of any one of claims 1 to 3, wherein the AAV rep and AAV cap genes are from different serotypes. Embodiment 5: The method of any one of claims 1 to 3, wherein the AAV rep and AAV cap genes are from the same serotype. Embodiment 6: The method of any one of claims 1 to 5, wherein the AAV rep gene is the AAV2 rep gene and the AAV cap gene is the AAV8 cap gene. Embodiment 7: The method of any one of claims 1 to 6, wherein the AAV ITRs and the AAV cap gene are from different serotypes. Embodiment 8: The method of any one of claims 1 to 6, wherein the AAV ITRs and the AAV cap gene are from the same serotype. Embodiment 9: The method of any one of claims 1 to 8, wherein the AAV inverted terminal repeat (ITR) sequence is an adeno-associated virus 2 inverted terminal repeat (ITR) sequence. Embodiment 10: The method of any one of claims 1 to 9, wherein the AAV ITR sequences are from the AAV2 serotype or a serotype selected from the group consisting of AAV1, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13. Embodiment 11: The method of any one of claims 1 to 10, wherein the AAV ITR sequences are synthetic. Aspect 12: 1 x 10 6 the total amount of nucleic acid transfected from (a), (b), and (c) per cell is less than 2 μg, and optionally, is less than 1×10 6 12. The method of claim 1, wherein the total amount of nucleic acid transfected from (a), (b) and (c) per cell is less than 1 Eg. Embodiment 13: The method of any one of claims 1-12, wherein the ratio of (a):(b):(c) is about 0.5-1.75: about 0.75-2.25: about 0.5-1.75 (wt:wt:wt), and optionally, the ratio of (a):(b):(c) is about 1: about 1-1.6: about 1 (wt:wt:wt). Embodiment 14: The method of any one of claims 1-13, wherein (a), (b), and (c) are transfected using a transfection composition comprising (a), (b), and (c) and a stable cationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is from about 1:1 to about 3:1 (weight / weight), and optionally, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1.5:1. Embodiment 15: The method of any one of claims 1 to 14, wherein each of (a), (b) and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules. Embodiment 16: A method according to any one of claims 1 to 15, wherein the nucleic acids (a), (b) and (c) to be transfected are synthetic nucleic acids and are free of eukaryotic and prokaryotic DNA modifications. Embodiment 17: The method of any one of claims 1-16, wherein the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding adenovirus helper (Ad helper) proteins, and optionally, wherein the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding adenovirus helper proteins E2A and E4. Aspect 18: The titer of the rAAV is at least 9.3 x 10 13 Vector genomes / 3.0×10 9 The method according to any one of claims 1 to 17, wherein the transfected cells are live transfected cells. Embodiment 19: A method according to any one of claims 1 to 18, wherein the suspension of the human embryonic cell line is cultured progressively in increasing volumes prior to transfection. Embodiment 20: The method of any one of claims 1 to 19, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 2000 liters. Aspect 21: The method of any one of claims 1 to 20, wherein the amino acid is included in the culture volume at a concentration of about 1 mM to about 20 mM. Embodiment 22: The method of any one of claims 1 to 21, wherein the culture medium having a volume of about 5 liters comprises the amino acid at a concentration of about 10 mM. Embodiment 23: The method of claim 21 or 22, wherein the amino acid is L-glutamine or L-alanyl-L-glutamine (Glutamax™). Embodiment 21: The method of any one of claims 1 to 24, wherein the culture medium having a volume of about 50 liters comprises at least about 1 mM to about 20 mM L-glutamine, at least about 0.01% to about 1% non-ionic surfactant polyol or detergent, and at least about 0.001% to about 1% antifoam agent. Aspect 24: The method of claim 24, wherein the nonionic surfactant polyol comprises pluronic acid. Aspect 26: A cultured human embryonic cell line containing approximately 3.0 x 10 viable cells 6 ~Approx. 1×10 8 The method of any one of claims 1 to 25, comprising a cell density of 1 / ml. Aspect 27: A cultured human embryonic cell line containing approximately 4.0 x 10 viable cells 6 From about 6 × 10 6 up to 2.5 x 10 cells / ml, or optionally up to 2.5 x 10 cells / ml 7 27. The method of any one of claims 1 to 26, comprising a cell density of up to cells / ml. Embodiment 28: The method of any one of claims 1-27, further comprising the steps of: (i) adding about 1 liter of media to the transfected cells; and (ii) adding a cationic polymer in a ratio of about 1:1 polymer to DNA to about 3:1 polymer to DNA over a time course of about 10 minutes to about 60 minutes. Embodiment 29: The method of claim 28, wherein the cationic polymer is added at a ratio of polymer to DNA of 2.2:1 over a time course of from about 1 minute to about 10 minutes. Embodiment 30: The method of any one of claims 14 to 29, wherein the cationic polymer comprises fully hydrolyzed linear polyethyleneimine (PEI). Embodiment 31: A method according to any one of claims 1 to 30, wherein the temperature of the culture medium containing the human embryonic cell suspension is raised to 37°C about 12 to 36 hours before transfection. 28. The method of claim 27, wherein the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM. 28. The method of claim 27, wherein the culture medium is subjected to air sparging at a flow rate of about 0.5 LPM. Embodiment 34: A method for producing a population of high titer recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, comprising: (i) transfecting a mammalian cell line with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication, (b) a nucleic acid sequence encoding rep and cap genes, and (c) at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, wherein the transfection comprises transfecting 1×10 cells / ml of the mammalian cell line with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication, (b) a nucleic acid sequence encoding rep and cap genes, and (c) at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, 6 the total amount of nucleic acid transfected from (a), (b) and (c) per cell is less than 2 μg, e.g., less than 1 μg; (ii) culturing the transfected cells for at least 24 hours, e.g., at least 40 hours; and (iii) harvesting the transfected cells and purifying the produced rAAV vector particles; and the titer of the rAAV is at least 9.3 x 10 13 Vector genomes / 3.0×10 9 The method, wherein the cells are live transfected cells. Embodiment 35: The method of claim 34, wherein the mammalian cell line is a suspension cell line and the cells are transfected in suspension. Embodiment 36: The method of any one of claims 34 or 35, wherein the cell line is derived from a human fetal kidney cell line. Embodiment 37: The method of any one of claims 34 to 36, wherein the mammalian cell line is a suspension-adapted serum-free cell line. Embodiment 38: The method of any one of claims 34 to 37, wherein the ratio of (a):(b):(c) is about 0.5 to 1.75: about 0.75 to 2.25: about 0.5 to 1.75 (weight:weight:weight), for example, the ratio of (a):(b):(c) is about 1: about 1 to 1.6: about 1 (weight:weight:weight). Embodiment 39: The method of any one of claims 34 to 38, wherein (a), (b), and (c) are transfected using a transfection composition comprising (a), (b), and (c) and a stable cationic polymer, and wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:1 to about 3:1 (weight / weight), e.g., the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1.5:1. Embodiment 40: The method of any one of claims 34 to 39, wherein each of (a), (b) and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules. Embodiment 41: A method according to any one of claims 34 to 40, wherein the nucleic acids (a), (b) and (c) to be transfected are synthetic nucleic acids and are free of eukaryotic and prokaryotic DNA modifications. Embodiment 42: The method of any one of claims 34 to 41, wherein (a) the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding Ad helper proteins, and optionally, the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding adenoviral helper proteins E2A and E4. Embodiment 43: The method of any one of claims 34 to 42, wherein the total amount of DNA from (a), (b) and (c) is, optionally, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.2, 1.4, 1.6 or 1.8 Eg. Embodiment 44: A method according to any one of claims 34 to 43, wherein the suspension of the mammalian cell line is cultured progressively in increasing volumes of culture medium before transfection. Embodiment 45: The method of any one of claims 34 to 44, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 2000 liters. Embodiment 46: The method of any one of claims 34 to 45, wherein an amino acid is included in the culture volume at a concentration of about 1 mM to about 20 mM. Embodiment 47: The method of any one of claims 34 to 46, wherein the culture medium having a volume of about 5 liters comprises the amino acid at a concentration of about 10 mM. Embodiment 48: The method of any one of claims 34 to 47, wherein the amino acid is L-glutamine. Embodiment 49: The method of any one of claims 34 to 48, wherein the cells are in a culture volume of 50 to 100 liters. Aspect 50: Infectious particle titer of at least 3 x 10 9 The method according to any one of claims 34 to 49, wherein the concentration is TCID50 / ml. Embodiment 51: The method of any one of claims 34 to 50, wherein the AAV Rep and AAV Cap genes are from the same AAV serotype. Embodiment 51: The method of any one of claims 34 to 50, wherein the AAV Rep and AAV Cap genes are from different AAV serotypes. Embodiment 53: The method of any one of claims 34 to 52, wherein the AAV ITRs and the AAV Cap gene are from the same AAV serotype. Embodiment 54: The method of any one of claims 34 to 52, wherein the AAV ITRs and the AAV Cap gene are from different AAV serotypes. Embodiment 55: The method of any one of claims 34 to 54, wherein the AAV ITR sequences are derived from AAV2 or a serotype selected from the group consisting of AAV1, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13. Embodiment 56: The method of any one of claims 34 to 55, wherein the AAV ITR sequences are synthetic. Embodiment 57: A method for producing a population of purified recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, comprising: (i) transfecting a mammalian cell line suspended in culture medium with a transfection composition, the transfection composition comprising: (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication; (b) a nucleic acid sequence encoding rep and cap genes; (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements; and (d) a stable cationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is at least 1:1, e.g., the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is at least 1.5:1; (ii) culturing the transfected cell line for at least 24 hours, e.g., at least 40 hours; (iii) harvesting the transfected cell line of step (ii); and (iii) Purifying the rAAV and purified virus was 2 x 10 4 The method has a particle to infectivity ratio of less than vg / TCID50. Embodiment 57: The method of claim 57, wherein the mammalian cell line is a suspension cell line and the cells are transfected in suspension. Embodiment 59: The method of any one of claims 57 to 58, wherein the mammalian cell line is derived from a human fetal kidney cell line. Embodiment 60: The method of any one of claims 57 to 59, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human fetal kidney cell line. Embodiment 61: The method of any one of claims 57 to 60, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b) and (c) is about 1.75:1 to about 2.75:1, for example, the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b) and (c) is about 2:1. Embodiment 62: The method of any one of claims 57 to 61, wherein the stable cationic polymer comprises fully hydrolyzed linear polyethyleneimine (PEI). Embodiment 63: The method of any one of claims 57 to 62, wherein the stable cationic polymer comprises fully hydrolyzed linear polyethyleneimine, and the ratio of PEI to nucleic acid is selected from the group consisting of 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.5:1, 2.8:1, and 2.2:1. Embodiment 64: A method according to any one of claims 57 to 63, wherein the temperature of the culture medium containing the cell suspension is raised to 37°C about 12 to 36 hours before transfection. Embodiment 65: The method of any one of claims 57 to 64, wherein the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM. Embodiment 66: The method of any one of claims 57 to 65, wherein the culture medium is subjected to air sparging at a flow rate of about 0.5 LPM. Embodiment 66: A method according to any one of claims 57 to 66, wherein the transfection composition is added to the suspension cells over a time course of from about 10 minutes to about 60 minutes. Embodiment 68: The method of any one of claims 57 to 67, wherein culture medium is added after step (i) and before step (ii). Embodiment 69: A method according to any one of claims 57 to 68, wherein the total amount of nucleic acid (DNA) from (a), (b) and (c) is from about 1 μg to about 20 μg. Embodiment 70: The method of any one of claims 57 to 69, wherein the total amount of DNA from (a), (b) and (c) is from about 1 μg to about 10 μg. Embodiment 71: The method of any one of claims 57 to 70, wherein the ratio of (a):(b):(c) is about 0.5 to 1.75: about 0.75 to 2.25: about 0.5 to 1.75 (weight:weight:weight). Embodiment 72: The method of any one of claims 57 to 71, wherein each of (a), (b) and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules. Embodiment 73: A method according to any one of claims 57 to 72, wherein the nucleic acids (a), (b) and (c) to be transfected are synthetic and free of eukaryotic and prokaryotic DNA modifications. Embodiment 74: The method of any one of claims 57 to 73, wherein the ratio of (a):(b):(c) is about 1:about 1 to 1.6:about 1 (wt:wt:wt). Embodiment 75: The method of any one of claims 57 to 74, wherein (a) the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises nucleotides encoding Ad helper proteins, and optionally, the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises nucleotide sequences encoding adenoviral helper proteins E2A and E4. Embodiment 76: The method of any one of claims 57 to 75, wherein the total amount of DNA from (a), (b) and (c) is 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6 or 1.8 μg. Embodiment 77: The method of any one of claims 57 to 76, wherein the total amount of DNA from (a), (b) and (c) is about 0.75 μg. Embodiment 78: A method according to any one of claims 57 to 77, wherein the suspension of the mammalian cell line is cultured progressively in increasing volumes prior to transfection. Embodiment 79: The method of any one of claims 57 to 78, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 2000 liters. Embodiment 80: The method of any one of claims 57 to 79, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 100 liters. Embodiment 81: The method of any one of claims 57 to 80, wherein an amino acid is included in the culture volume at a concentration of about 1 mM to about 20 mM. Embodiment 82: The method of any one of claims 57 to 81, wherein the culture medium having a volume of about 5 liters comprises an amino acid at a concentration of about 10 mM. Embodiment 83: The method of claim 81 or 82, wherein the amino acid is L-glutamine or L-alanyl-L-glutamine (Glutamax™). Embodiment 84: The method of any one of claims 57 to 83, wherein the cells are in a culture volume of between 50 liters and 100 liters. Embodiment 85: The method of any one of claims 57 to 84, wherein the culture medium having a volume of about 50 liters comprises at least about 1 mM to about 20 mM L-glutamine, at least about 0.01% to about 1% non-ionic surfactant polyol or detergent, and at least about 0.001% to about 1% antifoam agent. Embodiment 86: The method of claim 85, wherein the nonionic surfactant polyol comprises pluronic acid. Embodiment 87: The method of any one of claims 57 to 86, wherein the transfection composition comprises at least about 5% volume / volume (v / v) to about 20% v / v of culture medium. Embodiment 88: A method according to any one of claims 57 to 87, wherein the transfection composition comprises from about 1 liter to about 5 liters of culture medium. Embodiment 89: A method according to any one of claims 57 to 88, wherein the transfection composition comprises 5 to 50% (volume / volume) of culture medium. Embodiment 90: The nucleic acid sequence added to the transfection is 0.5 x 10 6 ~Approx. 5×10 6 90. The method of any one of claims 57 to 89, comprising about 0.1 μg to about 1 μg of Ad helper DNA, Rep / Cap DNA, or transgene per cell. Embodiment 91: The method of any one of claims 57 to 90, wherein the AAV Rep and AAV Cap genes are from the same AAV serotype. Embodiment 92: The method of any one of claims 57 to 90, wherein the AAV Rep and AAV Cap genes are from different AAV serotypes. Embodiment 93: The method of any one of claims 57 to 92, wherein the AAV ITRs and the AAV Cap gene are from the same AAV serotype. Embodiment 94: The method of any one of claims 57 to 92, wherein the AAV ITRs and the AAV Cap gene are from different AAV serotypes. Embodiment 95: The method of any one of claims 57 to 94, wherein the AAV ITR sequences are derived from AAV2 or a serotype selected from the group consisting of AAV1, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13. Embodiment 96: The method of any one of claims 57 to 95, wherein the Cap gene is derived from the AAV8 serotype. Embodiment 97: The method of any one of claims 1 to 96, wherein the packaged nucleic acid of the rAAV further lacks eukaryotic DNA sequences. Embodiment 98: The method of any one of claims 1 to 97, wherein the closed-ended linear double-stranded nucleic acid comprises one-half of a protelomerase binding site. Embodiment 99: The method of any one of claims 1 to 98, wherein the closed-ended linear double-stranded nucleic acid comprises one-half of a protelomerase binding site, wherein the one-half of the protelomerase binding site is formed by protelomerase digestion of a target binding site comprising a double-stranded palindromic sequence of at least 10 base pairs in length. Embodiment 100: A population of rAAV virions lacking prokaryotic DNA, produced by the method of any one of claims 1 to 99. Embodiment 101: A recombinant adeno-associated virus (rAAV) comprising a protelomerase target sequence. Embodiment 102: The rAAV of claim 101, wherein the protelomerase target sequence comprises a double-stranded palindromic sequence of at least 10 base pairs in length. Embodiment 103: The rAAV of claim 101 or 102, further comprising a transgene.

[0145] Further exemplary aspects of the present invention can be represented by the following numbered embodiments 1-74.

[0023] Embodiment 1: A method for producing a high-titer population of recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences, comprising the steps of: (i) inoculating a cell culture medium, optionally comprising cells of a host cell line, with (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acid sequences encoding AAV rep and AAV cap genes, (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements, and (d) optionally a polycationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is from about 1:1 to about 3:1 (wt / wt), and optionally, 1 x 10 6 (ii) incubating the inoculated cell culture medium for a period of time sufficient to produce rAAV; and (iii) purifying the rAAV, wherein optionally, the titer of the rAAV produced is greater than the titer of rAAV produced by cell culture medium inoculated with a corresponding amount of plasmid DNA (pDNA) containing a heterologous transgene. Embodiment 2: A method for producing a high-titer population of recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences, comprising the steps of: (i) transfecting cells of a host cell line in a culture medium with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising: (a) nucleic acid sequences encoding helper proteins sufficient for rAAV replication; (b) nucleic acid sequences encoding AAV rep and AAV cap genes; and (c) at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements; 6wherein the total amount of nucleic acid from (a), (b), and (c) per host cell is less than about 2 μg, or (ii) the host cells are transfected with a transfection composition comprising (a), (b), and (c), and a polycationic polymer, wherein the ratio of polycationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:1 to about 3:1 (weight / weight); (ii) incubating the transfected host cells for a period of time sufficient to produce rAAV; (iii) optionally, lysing the transfected host cells; and (iv) purifying the rAAV, wherein optionally the titer of the rAAV produced is higher than the titer of rAAV produced using a host cell line transfected with a corresponding amount of pDNA containing a heterologous transgene. Aspect 3: The viral titer of the rAAV is at least 9.3 x 10 13 Vector genomes / 3.0×10 9 3. The method of embodiment 1 or 2, wherein the transfected cells are live transfected cells. Aspect 4: The viral titer of the rAAV is at least 3.5 x 10 11 The method according to any one of aspects 1 to 3, wherein the concentration is 1000 ppm / ml. Aspect 5: Purified rAAV is 2 x 10 4 5. The method of any one of embodiments 1 to 4, wherein the method has a particle to infectivity ratio of less than vg / TCID50. Embodiment 6: The method according to any one of embodiments 1 to 5, wherein the incubating of the transfected host cell line is performed for at least about 24 hours. Embodiment 7: The method according to any one of embodiments 1 to 6, wherein the incubation of the transfected host cell line is carried out for about 40 hours to about 400 hours. Embodiment 8: The method of any one of embodiments 1 to 7, wherein the host cell line is contained in a cell culture volume of at least about 50 liters. Embodiment 9: The method of any one of embodiments 1 to 8, wherein the host cell line is contained in a cell culture volume of about 50 liters to about 100 liters. Aspect 10: 1 x 10 610. The method of any one of aspects 1 to 9, wherein the total amount of nucleic acid from (a), (b), and (c) per cell is less than about 1.5 μg. Aspect 11: 1 x 10 6 11. The method of any one of aspects 1 to 10, wherein the total amount of nucleic acid from (a), (b), and (c) per cell is less than about 1 μg. Aspect 12: 1 x 10 6 12. The method of any one of aspects 1 to 11, wherein the total amount of nucleic acid from (a), (b), and (c) per cell is less than about 0.75 μg. Aspect 13: 1 x 10 6 13. The method of any one of aspects 1 to 12, wherein the total amount of nucleic acid from (a), (b), and (c) per cell is at least about 0.25 μg. Aspect 14: 1 x 10 6 14. The method of any one of aspects 1 to 13, wherein the total amount of nucleic acid from (a), (b), and (c) per cell is at least about 0.5 μg. Embodiment 15: The method of any one of embodiments 1 to 14, wherein the ratio of nucleic acids (a):(b):(c) is about 0.5 to 1.75:about 0.75 to 2.25:about 0.5 to 1.75 (weight:weight:weight). Embodiment 16: The method of any one of embodiments 1 to 15, wherein the ratio of nucleic acids (a):(b):(c) is about 0.75-1.5:about 1-1.75:about 0.75-1.25 (weight:weight:weight). Embodiment 17: The method of any one of embodiments 1 to 16, wherein the ratio of the (a):(b):(c) nucleic acids is about 1.4:about 1.5:about 1 (wt:wt:wt). Embodiment 18: The method of any one of embodiments 1 to 17, wherein the polycationic polymer is polyethyleneimine (PEI). Embodiment 19: The method of any one of embodiments 1 to 18, wherein the polycationic polymer is linear polyethyleneimine. Embodiment 20: The method of any one of embodiments 1 to 19, wherein the stable cationic polymer is fully hydrolyzed polyethyleneimine. Embodiment 21: The method of any one of embodiments 1 to 20, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is from about 1.5:1 to about 2.75:1. Embodiment 22: The method of any one of embodiments 1 to 21, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is from about 1.9:1 to about 2.6:1. Aspect 23: 1 x 10 6 23. The method of any one of aspects 1 to 22, wherein the total amount of nucleic acid from (a), (b), and (c) per cell is about 0.55 μg to about 0.75 μg, and the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 2:1 to about 2.5:1. Embodiment 24: The method of any one of embodiments 1 to 23, wherein the host cell line is infected with a transfection composition volume that is between about 5% and about 20% (volume / volume) of the host cell line culture volume. Embodiment 25: The method of any one of embodiments 1 to 24, wherein the host cell line is infected with a transfection composition volume of about 7.5% to about 15% (volume / volume) of the host cell line culture volume. Embodiment 26: The method of any one of embodiments 1 to 25, wherein the transfection composition is added to the host cells over a time course of about 10 minutes to about 60 minutes. Embodiment 27: The method according to any one of embodiments 1 to 26, further comprising the step of culturing the host cell line for a period of time prior to transfection of the host cell line. Embodiment 28: The method of embodiment 27, wherein the step of culturing the host cell line comprises increasing the culture volume from about 50 ml to about 2000 liters. Embodiment 29: The method of embodiment 27 or 28, wherein the step of culturing the host cell line comprises increasing the culture volume to from about 50 ml to about 100 liters. Embodiment 30: The method according to any one of embodiments 1 to 29, wherein the temperature of the culture medium comprising the host cell line is raised to 37°C about 12 hours to 36 hours before transfection. Embodiment 31: The method of any one of embodiments 1 to 30, wherein the culture medium comprises the amino acid at a concentration of from about 1 mM to about 20 mM. Embodiment 32: The method of any one of embodiments 1 to 31, wherein the culture medium comprises an amino acid at a concentration of about 5 mM to about 15 mM. Embodiment 33: The method of any one of embodiments 1 to 32, wherein the culture medium comprises an amino acid at a concentration of from about 7.5 mM to about 12.5 mM. Embodiment 34: The method of any one of embodiments 31 to 33, wherein the amino acid is L-glutamine or a dipeptide containing L-glutamine. Embodiment 35: The method of embodiment 34, wherein the dipeptide comprising L-glutamine is L-alanyl-L-glutamine. Embodiment 36: The method of any one of embodiments 1 to 35, wherein the culture medium comprises the non-ionic surfactant polyol or detergent at a concentration of about 0.01% to about 1% (weight / volume). Embodiment 37: The method of embodiment 36, wherein the nonionic surfactant polyol comprises pluronic acid. Embodiment 38: The method of any one of embodiments 1 to 37, wherein the culture medium comprises an antifoaming agent at a concentration of about 0.001% to about 1% (weight / volume). Embodiment 39: The method of any one of embodiments 1 to 38, wherein the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM. Embodiment 40: The method of any one of embodiments 1 to 39, wherein the culture medium is subjected to air sparging at a flow rate of between about 0.25 LPM and about 0.75 LPM. Embodiment 41: The method of any one of embodiments 1 to 40, wherein the host cell line is a mammalian cell line. Embodiment 42: The method of any one of embodiments 1 to 41, wherein the host cell line is a human cell line. Embodiment 43: The method of any one of embodiments 1 to 42, wherein the host cell line is a human embryonic cell line. Embodiment 44: The method of any one of embodiments 1 to 43, wherein the host cell line is a human fetal kidney cell line. Embodiment 45: The method of any one of embodiments 1 to 44, wherein the host cell line is a serum-free cell line. Embodiment 46: The method of any one of embodiments 1 to 45, wherein the host cell line is suspension adapted. Embodiment 47: The method of any one of embodiments 1 to 46, wherein the host cell line is suspended in a culture medium. Embodiment 48: The method according to any one of embodiments 1 to 47, wherein the cells of the host cell line are transfected in suspension. Embodiment 49: The host cell line comprises about 3.0 x 10 viable cells 6 ~Approx. 1×10 8 The method of any one of aspects 1 to 48, comprising a cell density of 0.1% to 0.5% cells / ml. Embodiment 50: The host cell line comprises about 4.0 x 10 viable cells 6 ~about 6×10 6 50. The method of any one of aspects 1 to 49, comprising a cell density of 0.1% to 0.5% by weight / ml. Embodiment 51 : The host cell line comprises about 2.5 x 10 viable cells 7 51. The method of any one of aspects 1 to 50, comprising a cell density of 0.1% to 0.5% cells / ml. Embodiment 52: The method of any one of embodiments 1 to 51, wherein at least one of the nucleic acids (a) and (b) is comprised in a closed-ended linear double-stranded nucleic acid molecule. Embodiment 53: The method of any one of embodiments 1 to 52, wherein each of the nucleic acids (a) and (b) is independently comprised in a closed-ended linear double-stranded nucleic acid molecule. Embodiment 54: The method according to any one of embodiments 1 to 53, wherein the closed-ended linear double-stranded nucleic acid comprises one-half of a protelomerase binding site. Embodiment 55: The method of any one of embodiments 1 to 54, wherein the closed-ended linear double-stranded nucleic acid comprises one-half of a protelomerase binding site, wherein the one-half of the protelomerase binding site is formed by protelomerase digestion of the target binding site, and comprises a double-stranded palindromic sequence of at least 10 base pairs in length. Embodiment 56: The method of any one of embodiments 1 to 55, wherein the AAV rep and AAV cap genes are from the same serotype. Embodiment 57: The method of any one of embodiments 1 to 56, wherein the AAV rep and AAV cap genes are from different serotypes. Embodiment 58: The method of any one of embodiments 1 to 57, wherein the AAV ITR sequences and the AAV cap gene are from the same serotype. Embodiment 59: The method of any one of embodiments 1 to 58, wherein the AAV ITR sequences and the AAV cap gene are from different serotypes. Embodiment 60: The method of any one of embodiments 1 to 59, wherein the AAV ITR sequences and the AAV rep gene are from the same serotype. Embodiment 61: The method of any one of embodiments 1 to 60, wherein the AAV ITR sequences and the AAV rep gene are from different serotypes. Embodiment 62: The method of any one of embodiments 1 to 61, wherein the AAV rep gene is derived from a serotype selected from the group consisting of AAV1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13. Embodiment 63: The method of any one of embodiments 1 to 62, wherein the AAV rep gene is derived from a serotype selected from the group consisting of AAV2, 3a, 3b, 8, 9 and 10. Embodiment 64: The method of any one of embodiments 1 to 63, wherein the AAV cap gene is derived from a serotype selected from the group consisting of AAV1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13. Embodiment 65: The method of any one of embodiments 1 to 64, wherein the AAV cap gene is derived from a serotype selected from the group consisting of AAV2, 3a, 3b, 8, 9 and 10. Embodiment 66: The method of any one of embodiments 1 to 65, wherein the AAV ITR sequences are derived from a serotype independently selected from AAV1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13. Embodiment 67: The method of any one of embodiments 1 to 66, wherein the AAV ITR sequences are derived from a serotype independently selected from AAV2, 3a, 3b, 8, 9 and 10. Embodiment 68: The method of any one of embodiments 1 to 67, wherein the AAV ITR sequences are synthetic sequences. Embodiment 69: The method of any one of embodiments 1 to 68, wherein the nucleic acid sequence encoding a helper protein sufficient for rAAV replication comprises a nucleotide sequence encoding an adenovirus helper protein. Embodiment 70: The method of any one of embodiments 1 to 69, wherein the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding adenoviral helper proteins E2A and E4. Embodiment 71: The method according to any one of embodiments 1 to 70, wherein at least one of the transfected nucleic acids is a synthetic nucleic acid and is free of eukaryotic and prokaryotic DNA modifications. Embodiment 72: The method of any one of embodiments 1 to 71, wherein the rAAV further lacks eukaryotic DNA sequences. Embodiment 73: The method of any one of embodiments 1 to 72, wherein incubating for a period sufficient to produce rAAV is incubating for a period of at least 24 hours. Embodiment 74: A population of rAAV virions produced by the method of any one of embodiments 1 to 73.

[0146] It should be understood that one, more than one, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. While various embodiments of the present invention have been described above, it should be understood that they are presented by way of example only, and not limitation. In accordance with this disclosure, many modifications can be made to the disclosed embodiments without departing from the spirit or scope of the present invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.

[0147] definition For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values ​​used in the specification and claims are to be understood as being modified by the term "about," even though in all instances the term "about" may not be expressly stated along with that value, amount, or range. Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are not, and need not be, exact but may be approximate and / or, where desired, larger or smaller to reflect tolerances, conversion factors, rounding, measurement error, etc., as well as other factors known to those of ordinary skill in the art, depending on the desired properties sought to be obtained by the teachings of the present disclosure. For example, the term "about" in reference to a value can be meant to encompass variations of ±100% in some embodiments, ±50% in some other embodiments, ±20% in some other embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the indicated amount, as appropriate for practicing the disclosed methods or utilizing the disclosed compositions.

[0148] Additionally, the term "about" when used in connection with one or more numbers or numerical ranges should be understood to refer to all such numbers, inclusive, within the range, modifying that range by extending the boundaries above and below the numerical values ​​set forth. Recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (e.g., reference to 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range.

[0149] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms thereof unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or derivatives thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising." It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" is a reference to one or more proteins, including equivalents thereof known to those of skill in the art, and so forth.

[0150] As used herein, the terms "comprising," "comprise," or "comprised," and their derivatives, referring to defined or described elements of an object, composition, apparatus, method, process, system, etc., are meant to be inclusive or open-ended, allowing for additional elements, thereby indicating that the defined or described object, composition, apparatus, method, process, system, etc. includes the set forth elements—or equivalents, where appropriate—and that other elements may also be included and still be included within the scope / definition of the defined object, composition, apparatus, method, process, system, etc.

[0151] As used herein, the term "helper virus" or "contaminating helper virus" refers to a virus used to produce copies of a helper virus-dependent viral vector, e.g., adeno-associated virus, which does not have the ability to replicate by itself. The helper virus is used to co-infect cells with the viral vector and provides proteins necessary for replication of the viral vector genome. This term encompasses intact viral particles, empty capsids, viral DNA, etc. Helper viruses commonly used to produce rAAV particles include adenovirus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, and vaccinia virus.

[0152] Helper viruses include adenovirus (AV), herpes simplex virus (HSV), and existing systems for producing AAV in insect cells using baculovirus. It has also been proposed that papillomaviruses can also provide helper functions for AAV (see, for example, Hermonat et al., Molecular Therapy 9, S289-S290 (2004)). Helper viruses include any virus capable of achieving AAV replication. AV is a non-enveloped nuclear DNA virus with a double-stranded DNA genome of approximately 36 kb. AV provides the E1a, E1b55K, E2a, E4orf6, and VA genes, enabling AAV replication and encapsidation, thereby rescuing latent AAV proviruses within cells. HSV is a family of viruses with a relatively large double-stranded linear DNA genome encapsidated in an icosahedral capsid surrounded by a lipid bilayer envelope. HSV is infectious and highly contagious. The following HSV-1 replication proteins have been identified as required for AAV replication: the helicase / primase complex (UL5, UL8, and UL52) and the DNA-binding protein ICP8 encoded by the UL29 gene, together with other proteins that enhance helper function.

[0153] The term "non-adherent cell line" or "suspension cell line," as used herein, refers to a cell line that can survive in suspension culture without attaching to a surface (e.g., tissue culture plastic carriers or microcarriers). Adaptation to a non-adherent cell line is a lengthy process that requires passage in decreasing amounts of serum, thereby selecting for an irreversibly altered cell population. This cell line can be grown to higher densities than adherent conditions can achieve, and is therefore more suitable for cultivation on an industrial scale, for example, in a bioreactor setting or in stirred cultures.

[0154] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the context clearly indicates otherwise.

[0155] The term "promoter," as used herein, is defined as a DNA sequence recognized by a cell's synthetic machinery, or introduced synthetic machinery, required to initiate transcription of a particular polynucleotide sequence. A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to that promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded by or specified by a gene, causes the gene product to be produced in a cell substantially only when the cell is a cell of the tissue type corresponding to that promoter.

[0156] A "protelomerase" target sequence is any DNA sequence whose presence in a DNA template allows its conversion to a closed, linear DNA by the enzymatic activity of protelomerase. In other words, a protelomerase target sequence is required for the cleavage and religation of double-stranded DNA by protelomerase to form a covalently closed, linear DNA. Typically, a protelomerase target sequence includes any perfect palindrome, i.e., any double-stranded DNA sequence with dyad symmetry, also referred to herein as a perfect inverted repeat. The length of the perfect inverted repeat varies depending on the individual organism. In Borrelia burgdorferi, the perfect inverted repeat is 14 base pairs long. In various mesophilic bacteriophages, the perfect inverted repeat is 22 base pairs or longer. Also, in some instances, such as Escherichia coli N15, a central perfect inverted palindrome is adjacent to an inverted repeat sequence, i.e., it forms part of a larger imperfect inverted palindrome.

[0157] As used herein, the term "recombinant AAV (rAAV) vector" or "gene delivery vector" refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., viral DNA [vDNA]) packaged within an AAV capsid. Alternatively, in some contexts, the term "vector" may be used to refer to only the vector genome / vDNA.

[0158] A "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) containing one or more heterologous nucleotide sequences. rAAV vectors typically require only the 145-base terminal repeat (TR) in cis to produce virus. All other viral sequences are not essential and can be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, an rAAV vector genome will retain only the minimum TR sequence to maximize the size of the transgene that can be efficiently packaged by the vector. Sequences encoding structural and nonstructural proteins can be provided in trans (e.g., from a vector, e.g., a plasmid, or by stably integrating the sequences into a packaging cell). The rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence, a synthetic, or other parvovirus TR sequence), optionally two TRs (e.g., two AAV TRs), which are typically 5' and 3' flanked by, but not necessarily contiguous with, the heterologous nucleotide sequence. The TRs can be the same as or different from each other.

[0159] The term "terminal repeat" or "TR" includes any viral terminal repeat and synthetic sequences that form hairpin structures and function as inverted terminal repeats (ITRs), such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al. The capsid structure of autonomous parvoviruses and AAV is described in detail in Bernard N. Fields et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also the descriptions of the crystal structures of AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99: 10405-10), AAV4 (Padron et al., (2005) I. Virol. 79: 5047-58), AAV5 (Walters et al., (2004) I. Virol. 78: 3361-71), and CPV (Xie et al., (1996) I. Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251: 1456-64).

[0160] "AAV terminal repeats" or "AAV TRs" can be derived from any AAV, including, but not limited to, serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, or any other AAV now known or later discovered. AAV terminal repeats need not have wild-type terminal repeat sequences (e.g., the wild-type sequence can be altered by insertions, deletions, truncations, or missense mutations), so long as at least one of the terminal repeats mediates the desired function, e.g., replication, viral packaging, integration, and / or proviral rescue, of a functional TR. One of skill in the art will understand the selection of Rep proteins that are functional with respect to replication of the functional TR.

[0161] The term "transgene" is used herein to appropriately refer to a polynucleotide or nucleic acid intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, for example, a gene encoding a polypeptide or protein. Suitable transgenes, for example, for use in gene therapy, are well known to those skilled in the art. For example, the vectors described herein can deliver and be used to deliver transgenes, including but not limited to those described in U.S. Patent Nos. 6,547,099, 6,506,559, and 4,766,072, published U.S. applications Nos. 20020006664, 20030153519, and 20030139363, and published PCT applications WO 01 / 68836 and WO 03 / 010180, as well as miRNAs and other transgenes, for example, WO2017 / 152149, each of which is incorporated herein by reference in its entirety.

[0162] The term "tropism," as used herein, refers to the preferential entry of a virus into a particular cell or tissue, optionally followed by the intracellular expression (e.g., transcription and, optionally, translation) of sequences carried by the viral genome, e.g., in the case of a recombinant virus, expression of a heterologous nucleic acid of interest.

[0163] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild-type gene. This definition may also include, for example, "allelic," "splice," "interspecies," or "polymorphic" variants. Splice variants may have significant identity to a reference molecule but may typically have more or fewer polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or may be absent. Interspecies variants are polynucleotide sequences that differ from one species to another. Particularly useful in the present invention are variants of wild-type gene products. Variants may result from at least one mutation in the nucleic acid sequence, resulting in an altered mRNA or a polypeptide whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that result in variants are usually due to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0164] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.1, 2.2, 2.7, 3, 4, 5, 5.5, 5.75, 5.8, 5.85, 5.9, 5.95, 5.99, and 6. This applies regardless of the broadness of the range.

[0165] All documents mentioned herein are incorporated herein by reference. All references, patents, and published patent applications cited throughout this application, and the contents of their figures and sequence listings, are incorporated herein by reference for all purposes as if each individual publication or patent document were individually indicated. By citing various references herein, applicants do not acknowledge that any particular reference is "prior art" to the present invention. Aspects of the compositions and methods of the present invention are illustrated in the following examples. [Example]

[0166] The following non-limiting examples serve to illustrate selected aspects of the present invention and do not limit the scope of the invention as set forth in the claims. It will be understood that variations in the proportions of the components shown and substitutions for elements will be apparent to those skilled in the art and are within the scope of the aspects of the present invention.

[0167] Example 1: Production of AAV using closed linear (cl) DNA The purpose of this study was to evaluate large-scale production of rAAV.

[0168] material and method TCID50 assay: The infectious titer (TCID50) method is used to evaluate the in vitro AAV infectivity of pharmaceuticals in HeLa RC32 cells. In this assay, HeLa RC32 cells are transduced with adenovirus type 5 helper virus and serial dilutions of the pharmaceutical. After 3 days of infection, cells are treated with proteinase K to digest proteins, and replicated AAV vector DNA is quantified using qPCR technology. This method utilizes DNA primers and a fluorescent dye-based detection system. The absolute amount of ITR target sequences from the vector DNA is interpolated from a standard curve generated using a plasmid. A test sample containing ITRs is prepared and used as an assay control. Results are expressed as infectious units per milliliter (IU / mL). Note that for comparison of TCID50 / mL between different preparations, TCID50 / mL is preferably normalized to vg / mL.

[0169] Table 1. Description of analytical tests and details to be achieved for 50 L scale vector production. TIFF2026032042000014.tif90147

[0170] The PRO10™ cell line (AskBio, NC, USA) used to produce recombinant adeno-associated viral vectors (rAAV) is a suspension-adapted, serum-free cell line derived from the human embryonic kidney cell line 293 (HEK293). PRO10™ viral vector production is a batch process performed at medium to high cell densities and utilizes a triple transfection method through condensation of linear polyethylenimine MAX with the required plasmid (pDNA) or closed linear (cl) DNA substrate in a production medium cocktail. Both the cell growth medium and the production medium are chemically defined and free of animal-derived components. Each DNA molecule provides key elements in recombinant AAV production. The first provides adenovirus helper (Ad helper) proteins for efficient replication and packaging of the vector, but lacks the adenovirus structural and replication genes essential for generating adenovirus. The second construct is an AAV8 or AAVrh10 transfection construct (packaging construct) containing the AAV2 rep gene and the AAV8 or AAVrh10 capsid (cap) protein gene. The third construct is an AAV vector construct encoding a therapeutic transgene, containing adeno-associated virus 2 inverted terminal repeat (ITR) sequences flanking (5' to 3') the gene of interest. The construct used in all experiments was a dual reporter of GFP and luciferase. In addition, in subsequent studies, two therapeutic transgene cassettes containing CYP and GAA transgenes were used.

[0171] Initial experiments were performed at bench scale (31.25 mL to 2 L) using a traditional nonblocking approach with design of experiments (DoE) to identify and optimize critical parameters related to production by simultaneously testing factors such as cDNA concentration and the ratio of cDNA to transfection reagent. All small-scale experiments were controlled by parallel vector production using an optimized triple plasmid transfection system. Additional factors that could be evaluated include, but are not limited to, medium, cell density, transfection time, transfection volume, temperature, and other cell-dependent or cell-independent factors.

[0172] Small-scale transfected cultures were incubated for approximately 72 hours post-transfection (hpt) and then harvested by mechanical cell lysis. Total vector production was assessed through vector genome (vg) quantification using an in-house qPCR-based DNase-resistant particle (DRP) method specific for the viral ITRs. Yields were typically 4-6 x 10 as indicated by qPCR. 11 Yields were further assessed by monitoring total viral particles (capsids) per mL (vp / mL) via transgene-targeted qPCR and ELISA. Relative packaging efficiency was also modeled by monitoring the A260 / 280 ratio at harvest of affinity-purified lysates via SEC-HPLC.

[0173] The primary goal of the small-scale screening experiment was to identify near-optimal transfection conditions for the 50 L scale-up portion of this experimental design. In both pDNA and clDNA experiments, cells were thawed, cultured, and progressively expanded to inoculate a 50 L production bioreactor. This cell culture expansion process continued in the production bioreactor until transient transfection. The transfected cell culture was incubated in the production bioreactor for approximately 72 hpt. At harvest, the transfected cell culture was lysed and clarified through depth and membrane filtration, followed by purification. Purification consisted of capture chromatography, gradient ultracentrifugation, ion exchange chromatography, ultrafiltration / diafiltration (UF / DF), and 0.2 μm filtration steps. Table 3 provides characterization studies for the rAAV vectors produced by pDNA and clDNA, respectively.

[0174] Detailed process description for 50L SUB upstream operation To generate a 50 L batch, cells were thawed, cultured, and progressively expanded to inoculate a 50 L production bioreactor. This cell culture expansion process continued in the production bioreactor until transient transfection. Here, the seed train growth medium was supplemented with L-glutamine to a final concentration of 10 mM, which was used to recover the frozen cell stock and expand the inoculum to a 5 L suspension using a 10 L WAVE bag bioreactor. The medium used in the WAVE suspension was supplemented with 0.2% PLURONIC™ acid. The growth medium used after inoculation of ThermoFisher 50L single-use stirred-tank bioreactors (SUB, STR) consists of seed train growth medium supplemented with approximately 1-100 mM GLUTAMAX™, approximately 0.01%-10% PLURONIC™ acid (ThermoFisher, Waltham, MA), and approximately 0.001%-1% FOAMAWAY™ (Gibco, Waltham, MA). GLUTAMAX™ is a stabilized dipeptide source of L-glutamine designed to prevent degradation and reduce the toxic generation of excess ammonia.

[0175] Transient transfection to produce AAV was performed on 3.25–4.25 × 10 viable cells via condensation of three clones of DNA with linear polyethyleneimine MAX (Polysciences Inc., Warrington, PA) (PEI MAX). 6 pieces / mL 3 The transfection cocktail was performed at a cell density of 1000 kJ / ml. The transfection cocktail comprised 10% (v / v) of the culture volume (5 L). Condensation was performed in a custom-made 10 L WAVE Rocker bag fitted with tubing compatible with a 50 L SUB. The transfection cocktail was first prepared by adding 4 L of medium to the rocker bag with gentle agitation (8° angle, 25 RPM) at 25°C. An air overlay of 0.2 LPM was performed to prevent the bag from collapsing. Plasmids (Table 2) were then added, followed by the addition of 1 L of medium.

[0176] Table 2. Ratio of each clDNA used normalized to cell density at the time of transfection. TIFF2026032042000015.tif40128

[0177] After the addition of medium, PEI was added over 1 minute, followed by the addition of 1 L of medium. The cocktail was incubated for 7 minutes and then transferred to a SUB. The transfected cell suspension was incubated for 3 hours and quenched with 10% (v / v) volume of chemically defined, serum-free HEK293 medium supplemented with 10 mM L-glutamine.

[0178] SUB control parameters The large-scale manufacturing platform utilized a Finesse G3Pro Universal Controller attached to a ThermoFisher jacketed 50L submersible. The single-use vessel was equipped with a three-blade, 45° pitch angle, axial impeller, and dual sparger (Frit-Drilled-Hole) configuration, along with a primary Finesse TruFluor pH / DO single-use probe sheath and a Pall Kleenpak connection for insertion of a secondary, reusable pH / DO probe. The day before media fill, the bag was installed and inflated with a 10 LPM air overlay. The optical / reusable DO probe was connected to the transmitter. On the day of fill, the DO probe was calibrated using a two-point slope calibration. After media addition, both the single-use and reusable pH probes were standardized using offline samples on a calibrated blood and gas analyzer.

[0179] The day before inoculation, the SUB temperature was increased to 37°C. The medium was then conditioned by saturating with a continuous drill-hole air sparge at a flow rate of 0.5 LPM (0.025 VVM). Prior to inoculation, both single-use and reusable DO probes were standardized to 100% air saturation using a one-point calibration.

[0180] After inoculation, the controller was set to supply a continuous drill-hole air sparge at 0.5 LPM, and the headspace was swept with 1 LPM of air overlay. DO was controlled through an O2 gas cascade, maintaining the set point by increasing the O2 flow rate to the fritted sparger from 0.00 to 5.00 LPM (0 to 100% DO output / 0 to 100% MFC-3 output). pH was controlled by increasing the CO2 gas flow to the fritted sparger from 0.00 to 2.00 LPM (0 to -100% output / 0 to 100% MFC-4 output) at the high end (7.0 to 14), but was allowed to naturally flow at the low end without using a base feed to control pH.

[0181] result 1×10 6 DoE assessment of total cDNA (μg) per viable cell and PEI:DNA ratio Under DoE settings, 1 × 10 6 The μg DNA per viable cell and PEI:DNA ratio were studied over a range of 0.5-2 μg and 1-3, respectively. The design was a unique response surface model (RSM) with three levels for each factor, allowing for the interpretation of both linear and quadratic effects. Overlapping center points within this design space were used to estimate the significance of each effect.

[0182] Total vector production at harvest was assessed via ITR-qPCR (Figure 1). The data show a 2-2.5-fold increase in specific (vg / cell) productivity when using clDNA as the starting material compared to pDNA as the starting material.

[0183] To identify significant and interacting factors and to discern optimal clDNA conditions for transfection, the plasmid values ​​were excluded and the response was modeled using a fit model. The results of the JMP analysis are summarized below.

[0184] (Table 3) Summary of fits TIFF2026032042000016.tif50128

[0185] (Table 4) Analysis of variance TIFF2026032042000017.tif41130

[0186] 1×10 6 A summary of the fits and analysis of variance for the factors μg clDNA per cell (total clDNA) and PEI:DNA ratio were analyzed (Figure 2). The regression model captures most of the variance observed in this experiment. R 2 The p value was 0.941, indicating that less than 6% of the variance in vg titer could be explained by factors such as total clDNA or PEI:DNA. Contour plots showed the effect of clDNA and PEI:DNA ratios on vector genome titers in cell lysates, expressed in vg / mL (Figure 3). As shown in Figure 4, the clDNA used to produce recombinant AAVrh10CYP suggests that less than 1 ug of DNA, e.g., 0.6–0.7 ug, is required to achieve high titers of AAV. Furthermore, Figures 5 and 6 show PEI:DNA ratios of 2.2 and 2.5 for producing recombinant AAVrh10CYP and AAV8GAA, respectively, with the optimal clDNA being 0.6 ug, considering both overall yield and packaging efficiency.

[0187] (Table 5) Estimated values ​​of parameters TIFF2026032042000018.tif77156

[0188] Implementation of 50L SUB For each 50 L lot, QC assays were performed on in-process samples, purified bulk and final product, and product release testing. The following QC assays were performed and the results are shown below:

[0189] Table 6. Analytical tests, details and results for pDNA and clDNA derived vectors TIFF2026032042000019.tif101138

[0190] Table 7. TCID50 / ml and particle-to-infectivity (vg / TCID50) ratio results for pDNA and clDNA-derived vectors TIFF2026032042000020.tif80146

[0191] As shown in Table 7, in the DoE experiments and in the 50L runs, the clDNA-derived vectors exhibited increased infectivity compared to the pDNA control, as indicated by the lower vg / TCID50 ratio in the clDNA compared to that of the pDNA.

[0192] conclusion Although the clDNA system has been shown to be usable for producing AAV, batch-to-batch consistency in small-scale manufacturing runs, along with linear scalability of vector production, needs to be demonstrated with other serotypes and transgene constructs, as was done with plasmid DNA. The secondary effect on total clDNA shown in the above experiments was also observed in similar experiments used to optimize the pDNA transfection process. Further experiments will reveal possible product-specific relationships for total clDNA and PEI, which need to be further evaluated. Further studies will be conducted to address variations between small- and large-scale production. These studies will focus on establishing the stability of the clDNA starting material, the handleability of clDNA prior to transfection, and evaluation of the PEI:clDNA ratio, the clDNA ratio, and the kinetics of PEI:clDNA complexation.

[0193] Apart from yield and strength, analytical test results from the 50 L scale experiments were consistent with each other. Assay results for purity, safety, quality, and identity were highly similar regardless of starting material.

[0194] Further studies are planned to understand the gaps in yield, packaging efficiency, and purity and potency in scaled-up vector preparations, along with further optimization work.

[0195] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention encompassed by the appended claims.

[0196] Sequence information SEQUENCE LISTING <110> ASKBIO, INC. <120> Recombinant AAV Production <150> US 62 / 962,911 <151> 2020-01-17 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (5)..(6) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (9)..(10) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (13)..(14) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (17)..(18) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (22)..(22) <223> a, c, t, g, unknown or other <400> 1 ncatnntann cgnntannat gn 22 <210> 2 <211> 22 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 2 ccattatacg cgcgtataat gg 22 <210> 3 <211> 22 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 3 gcatactacg cgcgtagtat gc 22 <210> 4 <211> 22 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 4 ccatactata cgtatagtat gg 22 <210> 5 <211> 22 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 5 gcatactata cgtatagtat gc 22 <210> 6 <211> 14 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 6 attatatata taat 14 <210> 7 <211> 24 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 7 ggcatactat acgtatagta tgcc 24 <210> 8 <211> 42 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 8 acctatttca gcatactacg cgcgtagtat gctgaaatag gt 42 <210> 9 <211> 90 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 9 cctatattgg gccacctatg tatgcacagt tcgcccatac tatacgtata gtatgggcga 60 actgtgcata cataggtggc ccaatatagg 90 <210> 10 <211> 56 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 10 tatcagcaca caattgccca ttatacgcgc gtataatgga ctattgtgtg ctgata 56 <210> 11 <211> 42 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 11 atgcgcgcat ccattatacg cgcgtataat ggcgataata ca 42 <210> 12 <211> 52 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 12 tagtcaccta tttcagcata ctacgcgcgt agtatgctga aataggttac tg 52 <210> 13 <211> 90 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 13 gggatcccgt tccatacata catgtatcca tgtggcatac tatacgtata gtatgccgat 60 gttacatatg gtatcattcg ggatcccgtt 90 <210> 14 <211> 38 <212> DNA <213> Unknown <220> <223> Description of Unknown: Target sequence <400> 14 tactaaataa atattatata tataattttt tattagta 38 <210> 15 <211> 54 <212> DNA <213> Unknown <220> <223> Description of Unknown: telRL target sequence <400> 15 tatcagcaca caattgccca ttatacgcgc gtataatgga ctattgtgtg ctga 54 <210> 16 <211> 42 <212> DNA <213> Unknown <220> <223> Description of Unknown: pal target sequence <400> 16 acctatttca gcatactacg cgcgtagtat gctgaaatag gt 42 <210> 17 <211> 22 <212> DNA <213> Unknown <220> <223> Description of Unknown: phiK02 telRL target sequence <400> 17 ccattatacg cgcgtataat gg 22 <210> 18 <211> 33 <212> DNA <213> Unknown <220> <223> Description of Unknown: loxP target sequence <400> 18 taacttcgta tagcatacat tatacgaagt tat 33 <210> 19 <211> 34 <212> DNA <213> Unknown <220> <223> Description of Unknown: FRT target sequence <400> 19 gaagttccta ttctctagaa agtataggaa cttc 34 <210> 20 <211> 100 <212> DNA <213> Unknown <220> <223> Description of Unknown: phiC31 attP target sequence <400> 20 cccaggtcag aagcggtttt cgggagtagt gccccaactg gggtaacctt tgagttctct 60 cagttggggg cgtagggtcg ccgacaygac acaaggggtt 100 <210> 21 <211> 100 <212> DNA <213> Unknown <220> <223> Description of Unknown: lambda attP target sequence <400> 21 tgatagtgac ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt 60 tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa 100

Claims

1. 1. A method for producing a recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences, comprising: Culturing a human embryonic cell line in suspension; transfecting a human embryonic cell line with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising: (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication; (b) a nucleic acid sequence encoding the AAV rep and AAV cap genes; and (c) at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements; Incubating the transfected human cell line for about 40 to 400 hours; and Optionally, lysing the transfected human cell line and purifying the nucleic acid sequence encoding the rAAV. thereby producing rAAV.

2. The method of claim 1, wherein the cells of the cell line are transfected in suspension.

3. 2. The method of claim 1, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human fetal kidney cell line.

4. The method of claim 1, wherein the AAV rep and AAV cap genes are from different serotypes.

5. The method of claim 1, wherein the AAV rep and AAV cap genes are from the same serotype.

6. 2. The method of claim 1, wherein the AAV rep gene is an AAV2 rep gene and the AAV cap gene is an AAV8 cap gene.

7. The method of claim 1, wherein the AAV ITRs and AAV cap genes are from different serotypes.

8. The method of claim 1, wherein the AAV ITRs and the AAV cap gene are from the same serotype.

9. 2. The method of claim 1, wherein the AAV inverted terminal repeat (ITR) sequence is an adeno-associated virus 2 inverted terminal repeat (ITR) sequence.

10. 2. The method of claim 1, wherein the AAV ITR sequences are derived from the AAV2 serotype or a serotype selected from the group consisting of AAV1, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13.

11. 2. The method of claim 1, wherein the AAV ITR sequences are synthetic.

12. 1×10 6 the total amount of nucleic acid transfected from (a), (b), and (c) per cell is less than 2 μg, and optionally, is less than 1×10 6 2. The method of claim 1, wherein the total amount of nucleic acid transfected from (a), (b) and (c) per cell is less than 1 μg.

13. 10. The method of claim 1, wherein the ratio of (a):(b):(c) is about 0.5-1.75: about 0.75-2.25: about 0.5-1.75 (wt:wt:wt), and optionally the ratio of (a):(b):(c) is about 1: about 1-1.6: about 1 (wt:wt:wt).

14. 2. The method of claim 1, wherein (a), (b), and (c) are transfected using a transfection composition comprising (a), (b), and (c) and a stable cationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:1 to about 3:1 (weight / weight), and optionally, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1.5:

1.

15. 2. The method of claim 1, wherein each of (a), (b) and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules.

16. 2. The method of claim 1, wherein the nucleic acids (a), (b) and (c) to be transfected are synthetic nucleic acids and are free of eukaryotic and prokaryotic DNA modifications.

17. The method of claim 1, wherein the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding adenovirus helper (Ad helper) proteins, and optionally, the nucleic acid sequence encoding helper proteins sufficient for rAAV replication comprises a nucleotide sequence encoding adenovirus helper proteins E2A and E4.

18. rAAV titer of at least 9.3 × 10 13 Vector genomes / 3.0 x 10 9 2. The method of claim 1, wherein the cells are live transfected cells.

19. 10. The method of claim 1, wherein the suspension of human embryonic cell line is cultured in progressively increasing volumes prior to transfection.

20. 20. The method of claim 19, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 2000 liters.

21. 21. The method of claim 20, wherein the amino acid is included in the culture volume at a concentration of about 1 mM to about 20 mM.

22. 22. The method of claim 21, wherein the culture medium having a volume of about 5 liters comprises the amino acid at a concentration of about 10 mM.

23. 22. The method of claim 21, wherein the amino acid is L-glutamine or L-alanyl-L-glutamine (Glutamax™).

24. 21. The method of claim 20, wherein the culture medium having a volume of about 50 liters comprises at least about 1 mM to about 20 mM L-glutamine, at least about 0.01% to about 1% of a non-ionic surfactant polyol or detergent, and at least about 0.001% to about 1% of an antifoam agent.

25. 25. The method of claim 24, wherein the nonionic surfactant polyol comprises pluronic acid.

26. The cultured human embryonic cell line contained approximately 3.0 × 10 viable cells. 6 ~Approx. 1×10 8 The method of claim 1, wherein the cell density is in cells / ml.

27. The cultured human embryonic cell line contained approximately 4.0 × 10 viable cells. 6 From about 6 × 10 6 up to 2.5 x 10 cells / ml, or optionally up to 2.5 x 10 cells / ml 7 27. The method of claim 26, comprising a cell density of up to 100 cells / ml.

28. 10. The method of claim 1, further comprising the steps of: (i) adding about 1 liter of media to the transfected cells; and (ii) adding a cationic polymer at a ratio of about 1:1 polymer to DNA to about 3:1 polymer to DNA over a time course of about 10 minutes to about 60 minutes.

29. 29. The method of claim 28, wherein the cationic polymer is added at a ratio of polymer to DNA of 2.2:1 over a time course of about 1 minute to about 10 minutes.

30. 29. The method of claim 28, wherein the cationic polymer comprises fully hydrolyzed linear polyethyleneimine (PEI).

31. 10. The method of claim 1, wherein the temperature of the culture medium containing the human embryonic cell suspension is raised to 37°C about 12 to 36 hours prior to transfection.

32. 28. The method of claim 27, wherein the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM.

33. 28. The method of claim 27, wherein the culture medium is subjected to air sparging at a flow rate of about 0.5 LPM.

34. 1. A method for producing a high-titer population of recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences, comprising: (i) transfecting a mammalian cell line with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication, (b) a nucleic acid sequence encoding rep and cap genes, and (c) at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, wherein the transfection comprises transfecting 1×10 cells / ml of the mammalian cell line with a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication, (b) a nucleic acid sequence encoding rep and cap genes, and (c) at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, 6 the total amount of nucleic acid transfected from (a), (b) and (c) per cell is less than 1 μg; (ii) culturing the transfected cells for at least 24 hours; (iii) harvesting the transfected cells and purifying the produced rAAV vector particles; and the titer of the rAAV is at least 9.3 x 10 13 Vector genomes / 3.0 x 10 9 The method, wherein the cells are live transfected cells.

35. 35. The method of claim 34, wherein the mammalian cell line is a suspension cell line and the cells are transfected in suspension.

36. 35. The method of claim 34, wherein the cell line is derived from a human fetal kidney cell line.

37. 37. The method of any one of claims 34 to 36, wherein the mammalian cell line is a suspension-adapted serum-free cell line.

38. 35. The method of claim 34, wherein the ratio of (a):(b):(c) is about 0.5-1.75: about 0.75-2.25: about 0.5-1.75 (wt:wt:wt), and optionally the ratio of (a):(b):(c) is about 1: about 1-1.6: about 1 (wt:wt:wt).

39. 35. The method of claim 34, wherein (a), (b), and (c) are transfected using a transfection composition comprising (a), (b), and (c) and a stable cationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1:1 to about 3:1 (weight / weight), and optionally, the ratio of the stable cationic polymer to the total amount of nucleic acid from (a), (b), and (c) is about 1.5:

1.

40. 35. The method of claim 34, wherein each of (a), (b) and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules.

41. 35. The method of claim 34, wherein the transfected nucleic acids (a), (b) and (c) are synthetic nucleic acids and are free of eukaryotic and prokaryotic DNA modifications.

42. The method of claim 34, wherein (a) the nucleic acid sequence encoding a helper protein sufficient for rAAV replication comprises a nucleotide sequence encoding an Ad helper protein, and optionally, the nucleic acid sequence encoding a helper protein sufficient for rAAV replication comprises a nucleotide sequence encoding adenovirus helper proteins E2A and E4.

43. 43. The method of any one of claims 34-42, wherein the total amount of DNA from (a), (b) and (c) is optionally 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.2, 1.4, 1.6 or 1.8 μg.

44. 44. The method of any one of claims 34 to 43, wherein the suspension of the mammalian cell line is cultured progressively in increasing volumes of culture medium prior to transfection.

45. 45. The method of claim 44, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 2000 liters.

46. 46. ​​The method of claim 45, wherein the amino acid is included in the culture volume at a concentration of about 1 mM to about 20 mM.

47. 47. The method of claim 46, wherein the culture medium having a volume of about 5 liters comprises the amino acid at a concentration of about 10 mM.

48. 48. The method of claim 47, wherein the amino acid is L-glutamine.

49. 49. The method of claim 48, wherein the cells are in a culture volume of 50 to 100 liters.

50. Infectious particle titer of at least 3 × 10 9 TCID50 / ml.

51. 35. The method of claim 34, wherein the AAV Rep and AAV Cap genes are from the same AAV serotype.

52. 35. The method of claim 34, wherein the AAV Rep and AAV Cap genes are from different AAV serotypes.

53. 35. The method of claim 34, wherein the AAV ITRs and the AAV Cap gene are from the same AAV serotype.

54. 35. The method of claim 34, wherein the AAV ITRs and AAV Cap genes are from different AAV serotypes.

55. 35. The method of claim 34, wherein the AAV ITR sequences are from AAV2 or a serotype selected from the group consisting of AAV1, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13.

56. 35. The method of claim 34, wherein the AAV ITR sequences are synthetic.

57. 1. A method for producing a population of purified recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, comprising: i. transfecting a mammalian cell line suspended in culture medium with a transfection composition, the transfection composition comprising: (a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication; (b) a nucleic acid sequence encoding rep and cap genes; and (c) a closed-ended, linear, double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements; and (d) a stable cationic polymer, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is at least 1:1, and optionally, the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is at least 1.5:1; ii. culturing the transfected cell line for at least 24 hours; iii. harvesting the transfected cell line of step (ii); iv. Purifying the rAAV and purified virus was 2 x 10 4 The method has a particle to infectivity ratio of less than vg / TCID50.

58. 58. The method of claim 57, wherein the mammalian cell line is a suspension cell line and the cells are transfected in suspension.

59. 58. The method of claim 57, wherein the mammalian cell line is derived from a human fetal kidney cell line.

60. 60. The method of any one of claims 57 to 59, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human fetal kidney cell line.

61. 58. The method of claim 57, wherein the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is from about 1.75:1 to about 2.75:1, and optionally, the ratio of the stable cationic polymer to the total amount of nucleic acid components from (a), (b), and (c) is about 2:

1.

62. 62. The method of any one of claims 57-61, wherein the stable cationic polymer comprises fully hydrolyzed linear polyethyleneimine (PEI).

63. 63. The method of claim 62, wherein the ratio of PEI to nucleic acid is selected from the group consisting of 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.5:1, 2.8:1, and 2.2:

1.

64. 58. The method of claim 57, wherein the temperature of the culture medium containing the cell suspension is raised to 37°C about 12 to 36 hours prior to transfection.

65. 58. The method of claim 57, wherein the culture medium is subjected to air sparging at a flow rate of about 0.1 LPM to about 1.0 LPM.

66. 66. The method of claim 65, wherein the culture medium is subjected to air sparging at a flow rate of about 0.5 LPM.

67. 58. The method of claim 57, wherein the transfection composition is added to the suspension cells over a time course of about 10 minutes to about 60 minutes.

68. 58. The method of claim 57, wherein culture medium is added after step (i) and before step (ii).

69. 58. The method of claim 57, wherein the total amount of nucleic acid (DNA) from (a), (b), and (c) is from about 1 μg to about 20 μg.

70. 58. The method of claim 57, wherein the total amount of DNA from (a), (b), and (c) is from about 1 μg to about 10 μg.

71. 58. The method of claim 57, wherein the ratio of (a):(b):(c) is about 0.5-1.75:about 0.75-2.25:about 0.5-1.75 (wt:wt:wt).

72. 58. The method of claim 57, wherein each of (a), (b) and (c) is provided on one or more closed-ended linear double-stranded nucleic acid molecules.

73. 58. The method of claim 57, wherein the transfected nucleic acids (a), (b) and (c) are synthetic and free of eukaryotic and prokaryotic DNA modifications.

74. 58. The method of claim 57, wherein the ratio of (a):(b):(c) is about 1:about 1 to 1.6:about 1 (wt:wt:wt).

75. The method of claim 57, wherein (a) the nucleic acid sequence encoding a helper protein sufficient for rAAV replication comprises a nucleotide sequence encoding an Ad helper protein, and optionally, the nucleic acid sequence encoding a helper protein sufficient for rAAV replication comprises a nucleotide sequence encoding adenovirus helper proteins E2A and E4.

76. 58. The method of claim 57, wherein the total amount of DNA from (a), (b), and (c) is 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, or 1.8 μg.

77. 77. The method of claim 76, wherein the total amount of DNA from (a), (b) and (c) is about 0.75 μg.

78. 58. The method of claim 57, wherein the suspension of the mammalian cell line is cultured progressively in increasing volumes prior to transfection.

79. 58. The method of claim 57, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 2000 liters.

80. 58. The method of claim 57, wherein the culture volume is increased progressively from a volume of about 50 ml to a volume of about 100 liters.

81. 80. The method of claim 79, wherein the amino acid is included in the culture volume at a concentration of about 1 mM to about 20 mM.

82. 81. The method of claim 79 or 80, wherein the culture medium having a volume of about 5 liters comprises the amino acid at a concentration of about 10 mM.

83. 83. The method of claim 81 or 82, wherein the amino acid is L-glutamine.

84. 84. The method of claim 83, wherein the cells are in a culture volume of between 50 liters and 100 liters.

85. 58. The method of claim 57, wherein the culture medium having a volume of about 50 liters comprises at least about 1 mM to about 20 mM L-glutamine, at least about 0.01% to about 1% of a non-ionic surfactant polyol or detergent, and at least about 0.001% to about 1% of an antifoam agent.

86. 86. The method of claim 85, wherein the nonionic surfactant polyol comprises pluronic acid.

87. 58. The method of claim 57, wherein the transfection composition comprises at least about 5% volume / volume (v / v) to about 20% v / v of the culture medium.

88. 58. The method of claim 57, wherein the transfection composition comprises from about 1 liter to about 5 liters of culture medium.

89. 58. The method of claim 57, wherein the transfection composition comprises 5 to 50% (volume / volume) of culture medium.

90. The nucleic acid sequence added for transfection is 0.5 × 10 6 ~Approx. 5×10 6 58. The method of claim 57, comprising about 0.1 μg to about 1 μg of Ad helper DNA, Rep / Cap DNA, or transgene per cell.

91. The method of any one of claims 1, 34 or 57, wherein the packaged nucleic acid of the rAAV further lacks eukaryotic DNA sequences.

92. 58. The method of claim 57, wherein the AAV Rep and AAV Cap genes are from the same AAV serotype.

93. 58. The method of claim 57, wherein the AAV Rep and AAV Cap genes are from different AAV serotypes.

94. 58. The method of claim 57, wherein the AAV ITRs and the AAV Cap gene are from the same AAV serotype.

95. 58. The method of claim 57, wherein the AAV ITRs and AAV Cap genes are from different AAV serotypes.

96. 58. The method of claim 57, wherein the AAV ITR sequences are from AAV2 or a serotype selected from the group consisting of AAV1, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and 13.

97. 58. The method of claim 57, wherein the Cap gene is from the AAV8 serotype.

98. 58. The method of claim 57, wherein the closed-ended linear double-stranded nucleic acid comprises one-half of a protelomerase binding site.

99. 99. The method of claim 98, wherein one half of the protelomerase binding site is formed by protelomerase digestion of a target binding site comprising a double-stranded palindromic sequence at least 10 base pairs in length.

100. 10. A population of rAAV virions lacking prokaryotic DNA produced by the method of any one of claims 1, 34 or 57.

101. Recombinant adeno-associated virus (rAAV) containing a protelomerase targeting sequence.

102. The rAAV of claim 101, wherein the protelomerase target sequence comprises a double-stranded palindromic sequence at least 10 base pairs in length.

103. The rAAV of claim 101, further comprising a transgene.

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