Adeno-associated virus packaging system

Abstract

Provided herein is a dual vector transfection system for the production of recombinant adeno-associated virus (rAAV). The dual vector transfection system generally includes: (1) a first nucleic acid vector including a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence including a rAAV genome including a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector including a helper virus gene.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 202,817, filed June 25, 2021, No. 63 / 262,218, filed October 7, 2021, and No. 63 / 266,646, filed January 11, 2022, the entire disclosures of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety (the above ASCII copy, created on June 21, 2022, is named "HMW-043_SL.txt" and is 336,866 bytes in size). [Background technology]

[0003] Adeno-associated viruses (AAV) have unique characteristics that make them attractive as vectors to deliver foreign DNA to cells for gene therapy purposes. Commercial production of AAV generally uses either mammalian or insect cell systems. Commercially available mammalian cell-based AAV production systems typically involve the transfection of cells with three plasmids: a first plasmid containing sequences encoding AAV Rep and AAV capsid proteins; a second plasmid containing the AAV vector genome; and a third plasmid containing one or more helper virus genes (usually adenovirus or herpesvirus genes). Such three-plasmid AAV production systems, although effective, are complex to optimize, leading to high costs of goods associated with commercial AAV therapy.

[0004] Thus, there is a need in the art for improved AAV manufacturing systems that result in efficient AAV production but with reduced complexity and cost. Summary of the Invention

[0005] The present disclosure provides a dual vector transfection system for the production of recombinant adeno-associated virus (rAAV). The dual vector transfection system described herein generally includes: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene. In such a dual vector transfection system, the first nucleic acid vector and the second nucleic acid vector, together with a host producer cell, provide all the components required for AAV production. The dual vector transfection system disclosed herein has been found to provide increased rAAV productivity compared to conventional triple vector transfection systems. Furthermore, the specific organization of components in the dual vector transfection system described herein has been found to provide superior rAAV productivity over prior art dual vector transfection systems.

[0006] Thus, in one aspect, the disclosure provides a first nucleic acid vector comprising: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, wherein the first nucleic acid vector does not comprise helper virus genes.

[0007] In certain embodiments, the nucleic acid vector comprises, in the 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, wherein the nucleic acid vector does not comprise helper virus genes.

[0008] In certain embodiments, the nucleic acid vector comprises, in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, wherein the nucleic acid vector does not comprise a helper virus gene, and the transgene is not selected from the group consisting of phenylalanine hydroxylase (PAH), arylsulfatase A (ARSA), iduronate 2-sulfatase (I2S), and an anti-complement component 5 (C5) antibody.

[0009] In certain embodiments, the nucleic acid vector comprises, in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, wherein the nucleic acid vector does not comprise helper virus genes, and wherein the AAV capsid protein does not comprise an amino acid that is at least 95% identical to an amino acid sequence from amino acid 203 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, and wherein the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G or Y, the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C, or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.

[0010] In certain embodiments, the nucleic acid vector comprises, in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, wherein the nucleic acid vector does not comprise a helper virus gene, and (i) the transgene is not selected from the group consisting of phenylalanine hydroxylase (PAH), arylsulfatase A (ARSA), iduronate 2-sulfatase (I2S), and anti-complement component 5 (C5) antibody, and (ii) the AAV capsid protein is not encoded by a nucleotide sequence encoding an AAV Rep protein. The protein does not contain an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, and the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G or Y, the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C, or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.

[0011] In certain embodiments, the nucleic acid vector comprises, in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein.

[0012] In certain embodiments, the nucleic acid vector is a DNA plasmid or a DNA minimal vector.

[0013] In another aspect, the disclosure provides a recombinant AAV (rAAV) packaging system comprising: (i) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein; and (ii) a second nucleic acid vector comprising helper virus genes.

[0014] In certain embodiments, the first nucleic acid vector comprises, in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein. In certain embodiments, the transgene is not selected from the group consisting of phenylalanine hydroxylase (PAH), arylsulfatase A (ARSA), iduronate 2-sulfatase (I2S), and anti-complement component 5 (C5) antibody. In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, and includes any of the following: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.In certain embodiments, the transgene is not selected from the group consisting of phenylalanine hydroxylase (PAH), arylsulfatase A (ARSA), iduronate 2-sulfatase (I2S), and anti-complement component 5 (C5) antibody, and the AAV capsid protein does not comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, and is selected from the group consisting of: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C, or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.

[0015] In certain embodiments, the first nucleic acid vector is a DNA plasmid or a DNA minimal vector. In certain embodiments, the second nucleic acid vector is a DNA plasmid or a DNA minimal vector.

[0016] In certain embodiments, the transgene encodes a polypeptide. In certain embodiments, the transgene encodes a miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, lncRNA, ribozyme or mRNA. In certain embodiments, the transgene encodes a protein selected from the group consisting of phenylalanine hydroxylase (PAH), glucose-6-phosphatase (G6Pase), iduronate-2-sulfatase (I2S), arylsulfatase A (ARSA), and frataxin (FXN). In certain embodiments, the transgene encodes glucose-6-phosphatase (G6Pase) or frataxin (FXN).

[0017] In certain embodiments, the rAAV genome further comprises a transcriptional regulatory element operably linked to the transgene. In certain embodiments, the transcriptional regulatory element comprises a promoter element and / or an intron element.

[0018] In certain embodiments, the rAAV genome further comprises a polyadenylation sequence. In certain embodiments, the polyadenylation sequence is 3' to the transgene.

[0019] In certain embodiments, the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:71, 85, 86, 87, or 88.

[0020] In certain embodiments, the rAAV genome further comprises a 5' inverted terminal repeat (5'ITR) nucleotide sequence 5' of the transgene and a 3' inverted terminal repeat (3'ITR) nucleotide sequence 3' of the transgene. In certain embodiments, the 5'ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39, 41 or 42, and / or the 3'ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 40, 43 or 44.

[0021] In certain embodiments, the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:75, 78, 80, 82 or 84.

[0022] In certain embodiments, the AAV Rep protein is a wild-type Rep protein or a variant thereof. In certain embodiments, the AAV Rep protein is an AAV2 Rep protein or a variant thereof.

[0023] In certain embodiments, the first nucleotide sequence further comprises a transcriptional regulatory element operably linked to the AAV Rep protein coding sequence. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a native promoter. In certain embodiments, the promoter is selected from the group consisting of a P5 promoter, a P19 promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter, and a rapamycin-inducible promoter.

[0024] In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVRh32.33, AAVrh74, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, and PHP.S. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhlO, and AAVrh74. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8 and AAVrh74.

[0025] In certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0026] In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I; the amino acid in the capsid protein is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.

[0027] In certain embodiments, (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is H, the amino acid in the capsid protein corresponding to amino acid 682 of SEQ ID NO:16 is H, and the amino acid in the capsid protein corresponding to amino acid 683 of SEQ ID NO:16 is H. (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C.

[0028] In certain embodiments, the AAV capsid protein comprises the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0029] In certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 138 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0030] In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO:16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO:16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.

[0031] In certain embodiments, (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is H, (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C.

[0032] In certain embodiments, the AAV capsid protein comprises the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16 or 17.

[0033] In certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 1 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0034] In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO:16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO:16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO:16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO:16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO:16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of sequence number 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

[0035] In certain embodiments, (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y; (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (f) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is Y; (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; or the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C.

[0036] In certain embodiments, the AAV capsid protein comprises the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0037] In certain embodiments, the third nucleotide sequence further comprises a transcriptional regulatory element operably linked to the AAV capsid protein coding sequence. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a native promoter. In certain embodiments, the promoter is selected from the group consisting of a P40 promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter, and a rapamycin-inducible promoter.

[0038] In certain embodiments, the first nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:73 or 77.

[0039] In certain embodiments, the second nucleotide sequence comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:71, 75, 78, 80, 82, 84, 85, 86, 87, or 88.

[0040] In certain embodiments, the first nucleotide sequence comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:50, 51, 52, 53, 54, 55, 56, 57, 58, or 59; and the second nucleotide sequence comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:71, 75, 78, 80, 82, 84, 85, 86, 87, or 88. a third nucleotide sequence encoding an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of amino acids 203-736, 138-736 and / or 1-736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0041] In certain embodiments, the first nucleic acid vector comprises, in the 5' to 3' direction, a first nucleotide sequence; a second nucleotide sequence; and a third nucleotide sequence.

[0042] In certain embodiments, the helper virus gene is derived from a helper virus selected from the group consisting of adenovirus, herpesvirus, poxvirus, cytomegalovirus, and baculovirus. In certain embodiments, the helper virus gene is an RNA gene from an adenovirus selected from the group consisting of E1, E2, E4, and VA. In certain embodiments, the helper virus gene is a gene from a herpesvirus selected from the group consisting of UL5 / 8 / 52, ICP0, ICP4, ICP22, and UL30 / UL42.

[0043] In certain embodiments, the second nucleic acid vector further comprises a transcriptional regulatory element operably linked to the helper virus gene. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a native promoter. In certain embodiments, the promoter is selected from the group consisting of an RSV LTR promoter, a CMV immediate early promoter, an SV40 promoter, a dihydrofolate reductase promoter, a cytoplasmic β-actin promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter, and a rapamycin-inducible promoter.

[0044] In certain embodiments, the second nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:60, 61, or 62.

[0045] In certain embodiments, the second nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:63.

[0046] In another aspect, the disclosure provides a host cell comprising a nucleic acid vector described herein or a packaging system described herein. The disclosure also provides a population of such host cells. In certain embodiments, the population of host cells is provided in a cell culture. In certain embodiments, the cell culture has a volume of at least 2 liters, at least 50 liters, or at least 2000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 5000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 4000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 3000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 2500 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 2000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 1500 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 1000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 500 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 250 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 100 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 50 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 25 liters.

[0047] In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HEK293F cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, HeLa cells, NIH3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In certain embodiments, the mammalian cell is a HEK293 cell.

[0048] In another aspect, the disclosure provides methods for the recombinant preparation of rAAV comprising introducing a packaging system described herein into a mammalian cell under conditions that produce rAAV.

[0049] In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is selected from the group consisting of 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:3, or 1:4. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:2. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.2 to 1:1. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.6. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.8. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:1.

[0050] In certain embodiments, the method comprises introducing 0.1-4 μg DNA of the packaging system / 1E6 cells. In certain embodiments, the method comprises introducing 0.5-1 μg DNA of the packaging system / 1E6 cells. In certain embodiments, the method comprises introducing 0.6, 0.7, 0.8, 0.9, or 1 μg DNA of the packaging system / 1E6 cells. In certain embodiments, the method comprises introducing 0.75 μg DNA of the packaging system / 1E6 cells.

[0051] In certain embodiments, the ratio of the first nucleic acid vector to the second vector nucleic acid is 1:2, 1:3, or 1:4. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector is 1:2.

[0052] In certain embodiments, the method results in increased rAAV titer compared to a method comprising producing rAAV using a mammalian cell comprising: (i) a first vector comprising a nucleotide sequence encoding an AAV Rep protein and an AAV capsid protein; (ii) a second vector comprising an rAAV genome; and (iii) a third vector comprising one or more helper virus genes.

[0053] In certain embodiments, the method results in an increased percentage of intact vector genomes compared to a method comprising producing rAAV using a mammalian cell that contains (i) a first vector comprising a nucleotide sequence encoding AAV Rep proteins and AAV capsid proteins, (ii) a second vector comprising a rAAV genome, and (iii) a third vector comprising one or more helper virus genes.

[0054] In certain embodiments, the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HEK293F cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, HeLa cells, NIH3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In certain embodiments, the mammalian cell is a HEK293 cell. [Brief description of the drawings]

[0055] [Figure 1A] Graph showing viral genome (VG) productivity from small-scale rAAV production using a triple vector transfection system (1) and a dual vector transfection system (2). [Figure 1B]Graph showing capsid productivity obtained from small scale rAAV production using a triple vector transfection system (1) and a dual vector transfection system (2). [Figure 1C] FIG. 1 is a graph showing the percentage of intact vector genomes obtained from small-scale rAAV production using a triple vector transfection system (1) and a dual vector transfection system (2).

[0056] [Figure 2A] 1 is a graph showing VG productivity obtained from small scale rAAV production using a triple vector transfection system (1 and 3) and a dual vector transfection system (2 and 4). rAAV productivity was determined for two different rAAV gene editing vectors: a human specific gene editing vector (1 and 2) and a mouse specific vector (3 and 4). The different conditions are shown in Table 3. [Figure 2B] Graph showing capsid productivity obtained from small scale rAAV production using triple vector transfection system (1 and 3) and dual vector transfection system (2 and 4). rAAV productivity was determined for two different rAAV gene editing vectors: a human specific gene editing vector (1 and 2) and a mouse specific vector (3 and 4). The various conditions are shown in Table 3. [Figure 2C] Figure 1 is a graph showing the percentage of intact vector genomes obtained from small scale rAAV production using a triple vector transfection system (1 and 3) and a dual vector transfection system (2 and 4). rAAV productivity was determined for two different rAAV gene editing vectors: a human specific gene editing vector (1 and 2) and a mouse specific vector (3 and 4). The different conditions are shown in Table 3.

[0057] [Diagram 3]1A to 1C are schematic diagrams showing rAAV dual vector transfection systems Design-1 (A), Design-2 (B), and Design-3 (C).

[0058] [Figure 4A] 3A and 3B show the VG productivity from small-scale rAAV production using dual vector transfection system design-1 (1-3), dual vector transfection system design-2 (4-6), and triple vector transfection system (7). The dual vector transfection system designs tested are shown in Figures 3A and 3B. For each dual vector transfection system design tested, transfections were performed at three different transgene vector to helper vector ratios: 1:0.5 (1 and 4), 1:1 (2 and 5), and 1:3 (3 and 6). The various transfection conditions are listed in Table 4. [Figure 4B] Graphs showing capsid productivity from small-scale rAAV production using dual vector transfection system design-1 (1-3), dual vector transfection system design-2 (4-6), and triple vector transfection system (7). The dual vector transfection system designs tested are shown in Figures 3A and 3B. For each dual vector transfection system design tested, transfections were performed at three different transgene vector to helper vector ratios: 1:0.5 (1 and 4), 1:1 (2 and 5), and 1:3 (3 and 6). The various transfection conditions are listed in Table 4. [Figure 4C]Graph showing the percentage of intact vector genomes obtained from small-scale rAAV production using dual vector transfection system design-1 (1-3), dual vector transfection system design-2 (4-6), and triple vector transfection system (7). The dual vector transfection system designs tested are shown in Figures 3A and 3B. For each dual vector transfection system design tested, transfections were performed at three different transgene vector to helper vector ratios: 1:0.5 (1 and 4), 1:1 (2 and 5), and 1:3 (3 and 6). The various transfection conditions are listed in Table 4.

[0059] [Figure 5A] 3A-3C are graphs showing VG productivity from small-scale rAAV production using dual vector transfection system design-1 (1), dual vector transfection system design-2 (2), dual vector transfection system design-3 (3), and triple vector transfection system (4). The dual vector transfection system designs tested are shown in Figures 3A-3C. The various transfection conditions are listed in Table 5. [Figure 5B] Graphs showing capsid productivity from small-scale rAAV production using dual vector transfection system design-1 (1), dual vector transfection system design-2 (2), dual vector transfection system design-3 (3), and triple vector transfection system (4). The dual vector transfection system designs tested are shown in Figures 3A-3C. The various transfection conditions are listed in Table 5. [Figure 5C]Graph showing the percentage of intact vector genomes ("% complete") obtained from small-scale rAAV production using dual vector transfection system design-1 (1), dual vector transfection system design-2 (2), dual vector transfection system design-3 (3), and triple vector transfection system (4). The dual vector transfection system designs tested are shown in Figures 3A-3C. The various transfection conditions are listed in Table 5.

[0060] [Figure 6A] FIG. 1 is a graph showing VG productivity obtained from 2L scale rAAV production using Dual Vector Transfection System Design-1 at various transgene vector to helper vector ratios: 1:2 ("Dual 1:2"), 1:3 ("Dual 1:3"), and 1:4 ("Dual 1:4"), as well as the Triple Vector Transfection System (Triple): Six different rAAV vector genomes (1-6) were tested. Conditions 1-5 used the AAVHSC15 capsid and condition 6 used the AAVHSC17 capsid. The various transfection conditions are listed in Table 6. [Figure 6B] FIG. 1 is a graph showing capsid productivity obtained from 2L scale rAAV production using the Dual Vector Transfection System Design-1 at various transgene vector to helper vector ratios: 1:2 ("Dual 1:2"), 1:3 ("Dual 1:3"), and 1:4 ("Dual 1:4"), as well as the Triple Vector Transfection System (Triple): Six different rAAV vector genomes (1-6) were tested. Conditions 1-5 used AAVHSC15 capsids and condition 6 used AAVHSC17 capsids. The various transfection conditions are listed in Table 6. [Figure 6C]FIG. 1 is a graph showing the percentage of intact vector genomes obtained from 2L scale rAAV production using the Dual Vector Transfection System Design-1 at various transgene vector to helper vector ratios: 1:2 ("Dual 1:2"), 1:3 ("Dual 1:3"), and 1:4 ("Dual 1:4"), as well as the Triple Vector Transfection System (Triple): Six different rAAV vector genomes (1-6) were tested. Conditions 1-5 used the AAVHSC15 capsid and condition 6 used the AAVHSC17 capsid. The various transfection conditions are listed in Table 6.

[0061] [Figure 7A] VG productivity from small scale rAAV production using the Dual Vector Transfection System Design-1 (2TFX) and Triple Vector Transfection System (3TFX) utilizing AAV2 capsids. Various transfection conditions are listed in Table 6. [Figure 7B] 1 is a graph showing capsid productivity obtained from small scale rAAV production using the Dual Vector Transfection System Design-1 (2TFX) and Triple Vector Transfection System (3TFX) utilizing AAV2 capsids. The various transfection conditions are listed in Table 6. [Figure 7C] Graph showing the percentage of intact vector genomes obtained from small scale rAAV production using the Dual Vector Transfection System Design-1 (2TFX) and Triple Vector Transfection System (3TFX) utilizing the AAV2 capsid. The various transfection conditions are listed in Table 6.

[0062] [Figure 8]Figure 1 shows the number of intact vector genomes obtained from rAAV production using the Design-1 dual plasmid system, each expressed as a percentage increase over the number of intact vector genomes obtained from the corresponding triple plasmid system control. Four different rAAV vector genomes (1-4) were tested. Conditions 1-3 used the AAVHSC15 capsid and condition 4 used the AAVHSC17 capsid. The various transfection conditions are listed in Table 7.

[0063] [Figure 9] 1 is a graph showing the levels of capsid production from dual vector transfection systems Design-1 and Design-2, along with the levels of capsid production from vectors containing the Rep / Cap sequences of each design. The various transfection conditions are listed in Table 8.

[0064] [Figure 10A] 1 is a graph showing VG productivity from 50L bioreactor rAAV production using dual vector transfection system design-1 (2TFX) and triple vector transfection system (3TFX). Transfection conditions are listed in Table 6, condition 4, with a 1:2 vector ratio for design-1 and the associated triple transfection control. ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10B]Graph showing capsid productivity from 50L bioreactor rAAV production using dual vector transfection system design-1 (2TFX) and triple vector transfection system (3TFX). Transfection conditions are listed in Table 6, condition 4, with a 1:2 vector ratio for design-1 and the associated triple transfection control. ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10C] Figure 1 shows the percentage of intact vector genomes obtained from 50L bioreactor rAAV production using dual vector transfection system design-1 (2TFX) and triple vector transfection system (3TFX). Transfection conditions are listed in Table 6, condition 4, at a vector ratio of 1:2 for design-1 and with the associated triple transfection control. ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10D] Graph showing percent purity of residual host cell proteins in purified AAV vectors obtained using the 2TFX and 3TFX systems. ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10E] Graph showing percent aggregation in purified AAV vectors obtained using the 2TFX and 3TFX systems. ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10F]Graph showing residual host cell protein levels in purified AAV vectors obtained using the 2TFX and 3TFX systems. For assays where samples were determined to be below the limit of quantitation (BLoQ), the horizontal dashed line indicates the limit of detection. ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10G] Graph showing residual host cell DNA packaged into purified AAV vectors obtained using the 2TFX and 3TFX systems. ns means not significant; ns means not significant; * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10H] Graph showing the amount of Rep / Cap packaged into purified AAV vectors obtained using the 2TFX and 3TFX systems. ns means not significant. * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10I] Graph showing the amount of E1a packaged into purified AAV vectors obtained using the 2TFX and 3TFX systems. ns means not significant. The horizontal dashed line indicates the limit of detection for assays where samples were determined to be below the limit of quantitation (BLoQ). * means statistically significant at p<0.05; *** means statistically significant at p<0.001. [Figure 10J] Graph showing the amount of helper sequences packaged into purified AAV vectors obtained using the 2TFX and 3TFX systems. ns means not significant. * means statistically significant at p<0.05; *** means statistically significant at p<0.001.

[0065] [Figure 11A]Graph showing phenylalanine (Phe) concentrations measured in serum of Pahenu2 mice administered AAV vectors at a dose of 1E12VG / kg from condition 5, 1:4 vector ratio (design 1 (2TFX)) and the associated triple transfection control (3TFX) from Table 6. Vehicle only was administered as a control (vehicle). [Figure 11B] Graph showing phenylalanine (Phe) concentrations measured in serum of Pahenu2 mice administered AAV vectors at a dose of 1E14VG / kg from condition 5, 1:4 vector ratio (design 1 (2TFX)) and the associated triple transfection control (3TFX) from Table 6. Vehicle only was administered as a control (vehicle). [Figure 11C] FIG. 1 is a graph showing quantification of vector genomes in liver in treated mice at 6 weeks post-administration. ns means not significant. [Figure 11D] Graph showing quantification of transgene expression in treated mice 6 weeks after administration, ns means not significant. [Figure 11E] FIG. 13 is a graph showing quantification of on-target integration in treated mice at 6 weeks post-dosing. ns means not significant.

[0066] [Figure 12A] Figure 1 shows the VG productivity obtained from small scale rAAV production using the dual vector transfection system design-1, testing various ratios shown between vectors V3 and V12, at various levels of total DNA transfected (x-axis). The PEI:DNA ratio used was 2:1. [Figure 12B] Graph showing capsid productivity obtained from small scale rAAV production using dual vector transfection system design-1, testing various ratios shown between vectors V3 and V12, at various levels of total DNA transfected (x-axis). The PEI:DNA ratio used was 2:1. [Figure 12C] Figure 1 shows the percentage of intact vector genomes obtained from small scale rAAV production using the dual vector transfection system design-1, testing various ratios shown between vectors V3 and V12 at various levels of total DNA transfected (x-axis). The PEI:DNA ratio used was 2:1.

[0067] [Figure 13A] Figure 1 shows the VG productivity obtained from small scale rAAV production using the dual vector transfection system design-1, testing various ratios as shown between vectors V3 and V8, at various levels of total DNA transfected (x-axis). The PEI:DNA ratio used was 2:1. [Figure 13B] Figure 1 shows the capsid productivity obtained from small scale rAAV production using the dual vector transfection system Design-1, testing various ratios as shown between vectors V3 and V8, at various levels of total DNA transfected (x-axis). The PEI:DNA ratio used was 2:1. [Figure 13C] Figure 1 shows the percentage of intact vector genomes obtained from small scale rAAV production using the dual vector transfection system design-1, testing various ratios as shown between vectors V3 and V8 at various levels of total DNA transfected (x-axis). The PEI:DNA ratio used was 2:1.

[0068] [Figure 14A] 1 is a graph showing VG productivity obtained from 2L scale rAAV production using the dual vector transfection system design-1 and associated triple transfection controls spanning AAV capsid serotypes AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10 and AAVrh74. [Figure 14B]Graph showing capsid productivity obtained from 2L scale rAAV production using the dual vector transfection system design-1 and associated triple transfection controls spanning AAV capsid serotypes AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10 and AAVrh74. [Figure 14C] Graph showing the percentage of intact vector genomes obtained from 2L scale rAAV production using the dual vector transfection system design-1 and associated triple transfection controls spanning AAV capsid serotypes AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10 and AAVrh74.

[0069] [Figure 15] 1 is a graph showing VG productivity obtained from 50 L and 2000 L bioreactor rAAV production using the Dual Vector Transfection System Design-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] The present disclosure provides a dual vector transfection system for the production of recombinant adeno-associated virus (rAAV).The present disclosure is based on the finding that rAAV production using the dual vector transfection approach described herein results in superior AAV productivity compared to conventional triple vector transfection approach.The specific organization of components in the dual vector transfection system described herein also results in superior AAV productivity compared to conventional dual vector transfection approach.

[0071] I. Definition As used herein, the term "recombinant adeno-associated virus" or "rAAV" refers to an AAV that comprises a genome that lacks functional rep and cap genes.

[0072] As used herein, the term "cap gene" refers to a nucleic acid sequence that encodes an AAV capsid protein.

[0073] As used herein, the term "rep gene" refers to a nucleic acid sequence that encodes the AAV Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40) required for AAV replication.

[0074] As used herein, the term "Rep-Cap element" refers to a nucleic acid sequence encoding the AAV Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40) and AAV capsid proteins (e.g., VP1, VP2, and VP3) required for AAV replication.

[0075] As used herein, the term "helper virus genes" refers to nucleic acid sequences that encode viral genes (eg, adenovirus genes, or herpesvirus genes) that mediate AAV replication.

[0076] As used herein, the term "rAAV genome" refers to a nucleic acid molecule that contains the genomic sequence of an rAAV. Those skilled in the art will understand that when the rAAV genome contains a transgene, the rAAV genome can be in a sense or antisense orientation relative to the direction of transcription of the transgene.

[0077] As used herein, the term "edited genome" refers to a recombinant AAV genome that can incorporate an editing element (e.g., one or more nucleotides or internucleotide linkages) by homologous recombination into a target locus to correct a genetic defect in a target gene. One skilled in the art will understand that portions of the edited genome, including the 5' homology arm, the editing element, and the 3' homology arm, can be in a sense or antisense orientation relative to the target locus.

[0078] As used herein, the term "editing element" refers to a portion of an edited genome that modifies a target locus when integrated at the target locus. An editing element can mediate the insertion, deletion, or substitution of one or more nucleotides at a target locus. As used herein, the term "target locus" refers to a region of a chromosome or internucleotide bond (e.g., a region or internucleotide bond of a target gene) that is modified by an editing element.

[0079] As used herein, the term "homology arm" refers to a portion of the edited genome located 5' or 3' of an editing element that is substantially identical to the genome adjacent to the target locus.

[0080] As used herein, the "percentage of identity" between two nucleotide sequences or two amino acid sequences is calculated by multiplying the number of matches between the aligned sequence pairs by 100 and dividing this by the length of the aligned region, including internal gaps.Scoring identity only counts perfect matches and does not consider the similarity between amino acids.Please note that only internal gaps are included in the length, and gaps at the ends of sequences are not included.

[0081] As used herein, the term "coding sequence" refers to a portion of complementary DNA (cDNA) that encodes a polypeptide beginning with a start codon and ending with a stop codon. A gene may have one or more coding sequences due to alternative splicing, alternative translation initiation, and variation within a population. A coding sequence may be wild-type or a non-naturally occurring variant (e.g., a codon-optimized variant).

[0082] As used herein, the term "transcriptional regulatory element" or "TRE" refers to a cis-acting nucleotide sequence, e.g., a DNA sequence, that regulates (e.g., controls, increases, or decreases) transcription of an operably linked nucleotide sequence by an RNA polymerase to form an RNA molecule. A TRE depends on one or more trans-acting molecules, such as transcription factors, to regulate transcription. Thus, a single TRE may regulate transcription in different ways when contacted with different trans-acting molecules, e.g., in different types of cells. A TRE may contain one or more promoter elements and / or enhancer elements. One skilled in the art will understand that promoter and enhancer elements within a gene may be positionally close, and the term "promoter" may refer to a sequence that includes promoter and enhancer elements. Thus, the term "promoter" does not exclude enhancer elements in a sequence. The promoter and enhancer elements need not be derived from the same gene or species, and the sequence of each promoter or enhancer element may be identical or substantially identical to the corresponding endogenous sequence in the genome.

[0083] As used herein, the term "operably linked" is used to describe the link between a TRE and a coding sequence to be transcribed. Typically, gene expression is placed under the control of a TRE that includes one or more promoter and / or enhancer elements. A coding sequence is "operably linked" to a TRE if the transcription of the coding sequence is controlled or influenced by the TRE. The promoter and enhancer elements of the TRE may be in any orientation and / or distance from the coding sequence, so long as the desired transcriptional activity is obtained. In certain embodiments, the TRE is upstream of the coding sequence.

[0084] As used herein, the term "polyadenylation sequence" refers to a DNA sequence that constitutes a polyadenylation signal sequence when transcribed into RNA. The polyadenylation sequence may be natural or exogenous. The exogenous polyadenylation sequence may be a mammalian or viral polyadenylation sequence (e.g., SV40 polyadenylation sequence).

[0085] As used herein, "exogenous polyadenylation sequence" refers to a polyadenylation sequence that is not identical or substantially identical to the endogenous polyadenylation sequence of the transgene. In certain embodiments, the exogenous polyadenylation sequence is a polyadenylation sequence of a gene different from the transgene but within the same species (e.g., human). In certain embodiments, the exogenous polyadenylation sequence is a polyadenylation sequence of a different organism (e.g., a virus).

[0086] II. First Nucleic Acid Vector A conventional triple vector transfection system for producing rAAV typically includes a first vector containing sequences encoding AAV Rep protein and AAV capsid protein; a second vector containing the rAAV genome; and a third vector containing one or more helper virus genes. It has previously been shown that genes encoding AAV Rep protein, AAV capsid protein, and one or more helper virus genes can be cloned into the same vector ("Rep-Cap-Helper vector"). In such cases, double transfection of the Rep-Cap-Helper vector together with the second vector containing the rAAV genome (i.e., providing Rep, Cap, and helper genes in trans to the rAAV genome) can be used to produce rAAV. See, for example, Grimm et al. (1998) Hum. Gene Ther. 9(18):2745-2760, the disclosure of which is incorporated herein by reference in its entirety.

[0087] In contrast to previous dual vector transfection systems, the dual vector transfection system of the present disclosure provides Rep and Cap genes in cis with the rAAV genome. Thus, the present disclosure provides a dual vector transfection system for the production of recombinant adeno-associated virus (rAAV), and the dual vector transfection system described herein generally includes: (1) a first nucleic acid vector including a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence including a rAAV genome including a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector including a helper virus gene.

[0088] In certain embodiments, the first nucleic acid vector comprises, in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising an rAAV genome including a transgene; and a third nucleotide sequence encoding an AAV capsid protein. Certain aspects of the present disclosure provide that the first nucleic acid vector does not comprise helper virus genes (e.g., genes encoding AAV-produced helper factors).

[0089] The dual vector transfection system described herein generally involves transfecting a first nucleic acid vector and a second nucleic acid vector into a suitable host cell for producing AAV (e.g., rAAV). In certain embodiments, the first nucleic acid vector and the second nucleic acid vector together provide all the components necessary for AAV (e.g., rAAV) production. In certain embodiments, the first nucleic acid vector and the second nucleic acid vector, and further the host cell together provide all the components necessary for AAV (e.g., rAAV) production.

[0090] The dual vector transfection system disclosed herein has been found to result in increased rAAV productivity compared to both conventional triple vector transfection systems and the dual vector transfection systems described above. Without being bound by any theory, Applicants believe that providing the Rep and Cap genes in cis with the rAAV genome in the dual vector transfection system described herein results in superior rAAV productivity, in part, due to fewer empty AAV capsids being produced.

[0091] rAAV genome In the dual vector systems disclosed herein, the first nucleic acid vector generally comprises a nucleotide sequence comprising a rAAV genome. In certain embodiments, the rAAV genome comprises a transgene.

[0092] In certain embodiments, the transgene comprises one or more sequences encoding an RNA molecule. Suitable RNA molecules include, but are not limited to, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, mRNA, lncRNA, ribozyme, and synthetic RNA known in the art.

[0093] In certain embodiments, the transgene encodes one or more polypeptides or fragments thereof. Such transgenes may include the complete coding sequence of a polypeptide or may include only a fragment of the coding sequence of a polypeptide. In certain embodiments, the transgene encodes a polypeptide useful for treating a disease or disorder in a subject. Suitable polypeptides include, but are not limited to, β-globin, hemoglobin, tissue plasminogen activator, and clotting factors; colony-stimulating factor (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, and the like; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, e.g., basic FGF, and the like; and the like. and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma-derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), ), etc.; soluble receptors, such as soluble TNF-α receptor, soluble interleukin receptors (e.g., soluble IL-1 receptor and soluble type II IL-1 receptor), soluble gamma / delta T cell receptors, ligand-binding fragments of soluble receptors, etc.; enzymes, such as a-glucosidase, imiglucerase, beta-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP- 10. Monokine induced by interferon gamma (Mig), Groα / IL-8, RANTES, MIP-1a, MIP-1β, MCP-1, PF-4, etc.; angiogenic agents, such as vascular endothelial growth factor (VEGF, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2, etc.; angiogenesis inhibitors, such as soluble VEGF receptor; protein vaccines;Neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopeptide, Resin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); tissue factor; macrophage-activating factor; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); tissue inhibitor of metalloproteinases; vasoactive intestinal peptide; angiogenin; angio tropin; fibrin; hirudin; IL-1 receptor antagonist; ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophin 3 and 4 / 5 (NT-3 and -4 / 5); glial cell line-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); factor VIII, factor IX, factor X; dystrophin or mini-dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, β-enolase, glycogen synthase; lysosomal enzymes such as iduronate-2-sulfatase (I2S) and arylsulfatase A; and mitochondrial proteins such as frataxin.

[0094] In certain embodiments, the transgene encodes a protein that may be defective in one or more lysosomal storage diseases. Suitable proteins include, but are not limited to, α-sialidase, cathepsin A, α-mannosidase, β-mannosidase, glycosylasparaginase, α-fucosidase, α-N-acetylglucosaminidase, β-galactosidase, β-hexosaminidase α-subunit, β-hexosaminidase β-subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase A, saposin B, formylglycine generating enzyme, β-galactosylceramidase, α-galactosidase A, iduronate sulfatase, α-iduronidase, heparan N-sulfatase, acetyl-CoA transfer ... enzymes, N-acetylglucosaminidase, β-glucuronidase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, α-glucosidase, acid sphingomyelinase, acid ceramidase, acid lipase, cathepsin K, tripeptidyl peptidase, palmitoyl protein thioesterase, cystinosine, sialin, UDP-N-acetylglucosamine, phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NPC1, CLN3, CLN6, CLN8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit.

[0095] In certain embodiments, the transgene encodes an antibody or fragment thereof (e.g., a Fab, scFv, or full-length antibody). Suitable antibodies include, but are not limited to, muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, lamin ... Nibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, di Nutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, oviltoxiximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin , durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cemiplimab, fremanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab.

[0096] In certain embodiments, the transgene encodes a nuclease. Suitable nucleases include, but are not limited to, zinc finger nucleases (ZFNs) (see, e.g., Porteus, and Baltimore (2003) Science 300:763; Miller et al. (2007) Nat. Biotechnol. 25:778-785; Sander et al. (2011) Nature Methods 8:67-69; and Wood et al. (2011) Science 333:307, each of which is incorporated by reference in its entirety), transcription activator-like effector nucleases (TALENs) (see, e.g., Wood et al. (2011) Science 333:307; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1509-1512; 326:1501; Christian et al. (2010) Genetics 186:757-761; Miller et al. (2011) Nat. Biotechnol. 29:143-148; Zhang et al. (2011) Nat. Biotechnol. 29:149-153; and Reyon et al. (2012) Nat. Biotechnol. 30(5):460-465, each of which is incorporated by reference in its entirety), homing endonucleases, meganucleases (see, e.g., U.S. Patent Publication No. US2014 / 0121115, each of which is incorporated by reference in its entirety), and RNA-guided nucleases (see, e.g., Makarova et al. (2018) The CRISPR Journal 1(5):325-336; and Adli (2018) Nat. Communications 9:1911, each of which is incorporated by reference in its entirety.

[0097] In certain embodiments, the transgene encodes an RNA-guided nuclease. Suitable RNA-guided nucleases include, but are not limited to, class I and class II clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases. Class I is classified into types I, III, and IV, including, but not limited to, type I (Cas3), type IA (Cas8a, Cas5), type IB (Cas8b), type IC (Cas8c), type ID (Cas10d), type IE (Csel, Cse2), type IF (Csy1, Csy2, Csy3), type IU (GSU0054), type III (Cas10), type III-A (Csm2), type III-B (Cmr5), type III-C (Csx10 or Csx11), type III-D (Csx10), and type IV (Csf1). Class II is divided into types II, V, and VI, including, but not limited to, type II (Cas9), type II-A (Csn2), type II-B (Cas4), type V (Cpf1, C2c1, C2c3), and type VI (Cas13a, Cas13b, Cas13c). RNA-guided nucleases also include naturally occurring class II CRISPR nucleases such as Cas9 (type II) or Cas12a / Cpf1 (type V), as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).

[0098] In certain embodiments, the transgene encodes one or more reporter sequences that upon expression produce a detectable signal, including, but not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein (RFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane-bound proteins, such as CD2, CD4, CD8, influenza hemagglutinin protein, and others well known in the art (for which high affinity antibodies exist or can be produced by conventional means), as well as DNA sequences encoding fusion proteins that include membrane-bound proteins appropriately fused to antigen tag domains from hemagglutinin or Myc, among others.

[0099] In certain embodiments, the rAAV genome comprises a transcriptional regulatory element (TRE) operably linked to a transgene, which controls the expression of an RNA or polypeptide encoded by the transgene. In certain embodiments, the TRE comprises a constitutive promoter. In certain embodiments, the TRE can be active in any mammalian cell (e.g., any human cell). In certain embodiments, the TRE is active in a wide range of human cells. Such a TRE may comprise constitutive promoter and / or enhancer elements, including any of those described herein and any of those known to those of skill in the art. In certain embodiments, the TRE comprises an inducible promoter. In certain embodiments, the TRE may be a tissue-specific TRE, i.e., the TRE is active in a particular tissue(s) and / or organ(s). A tissue-specific TRE comprises one or more tissue-specific promoter and / or enhancer elements, and optionally one or more constitutive promoter and / or enhancer elements. One skilled in the art will appreciate that tissue-specific promoter and / or enhancer elements can be isolated from genes that are specifically expressed in a tissue by methods well known in the art.

[0100] Suitable promoters include, for example, the cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2):431-441), the CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2):269-277), the CB SB(Jacobson et al. (2006) Molecular Therapy 13(6):1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91(2):217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3):409-417), mitochondrial heavy chain promoter (Lodeiro et al. (2012) PNAS 109(17):6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261:101-105). In certain embodiments, the TRE is selected from the group consisting of a cytomegalovirus (CMV) promoter / enhancer (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18 or 19), an SV40 promoter, a chicken beta actin (CBA) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20 or 21), an smCBA promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:22), a human elongation factor 1 alpha ... alpha (EF1α) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:23), minute virus of mouse (MVM) intron containing transcription factor binding site (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24 or 25), human phosphoglycerate kinase (PGK1) promoter, human ubiquitin C (Ubc) promoter, human beta actin promoter, human neuron-specific enolase (ENO2) promoter, human beta glucuronidase (GUSB) promoter, rabbit beta-globin element (e.g.,26 or 27), human calmodulin 1 (CALM1) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28), human A poE / CI hepatic control region (HCR1) (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29), human alpha 1-antitrypsin (hAAT) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to SEQ ID NO:30, 31, or 32), , 99%, or 100% identical to SEQ ID NO:33), an extended HCR1 (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:33), an HS-CRM8 element of the hAAT promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:34), a human transthyretin (TTR) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:35), and / or a human methyl-CpG binding protein 2 (MeCP2) promoter. Any of the TREs described herein may be combined in any order to promote efficient transcription. For example, a rAAV genome may include a TRE (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36) that includes a CMV enhancer, a CBA promoter, and a splice acceptor from exon 3 of the rabbit beta-globin gene, collectively referred to as the CAG promoter ...A rAAV genome may include a TRE that includes a hybrid of the CMV enhancer and CBA promoter, followed by a splice donor and a splice acceptor, collectively referred to as the CASI promoter region (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37). For example, a rAAV genome may include a TRE that includes the HCR1 and hAAT promoters (also referred to as the LP1 promoter, e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38).

[0101] In certain embodiments, the TRE is brain-specific (e.g., neuron-specific, glial cell-specific, astrocyte-specific, oligodendrocyte-specific, microglia-specific, and / or central nervous system-specific). Exemplary brain-specific TREs may include, but are not limited to, one or more elements from the human glial fibrillary acidic protein (GFAP) promoter, the human synapsin 1 (SYN1) promoter, the human synapsin 2 (SYN2) promoter, the human metallothionein 3 (MT3) promoter, and / or the human proteolipid protein 1 (PLP1) promoter. Many more brain-specific promoter elements are disclosed in WO2016 / 100575A1, which is incorporated herein by reference in its entirety.

[0102] In certain embodiments, the native promoter for the transgene may be used.Natural promoters may be preferred when it is desired that the expression of the transgene mimics the native expression.Natural promoters may be used when it is necessary to regulate the expression of the transgene in a temporal or developmental manner, or in a tissue-specific manner, or in response to a specific transcriptional stimulus.In further embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic the native expression.

[0103] In certain embodiments, the rAAV genome comprises an edited genome. The edited genome can be used to edit the genome of a cell by homologous recombination of the edited genome with a genomic region surrounding the target locus in the cell. In certain embodiments, the edited genome is designed to correct a genetic defect of a gene by homologous recombination. The edited genome generally comprises: (i) an editing element for editing a target locus in a target gene; (ii) a 5' homology arm nucleotide sequence at the 5' of the editing element that has homology with a first genomic region that is 5' to the target locus; and (iii) a 3' homology arm nucleotide sequence at the 3' of the editing element that has homology with a second genomic region that is 3' to the target locus, where the portion of the edited genome that comprises the 5' homology arm, the editing element, and the 3' homology arm can be in a sense or antisense orientation with respect to the target locus. Target genes suitable for editing using an edited genome include, but are not limited to, phenylalanine hydroxylase (PAH), cystic fibrosis transmembrane conductance regulator (CFTR), beta hemoglobin (HBB), oculocutaneous albinism II (OCA2), huntingtin (HTT), myotonic dystrophy protein kinase (DMPK), low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulated endopeptidase homolog, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y).

[0104] In certain embodiments, the rAAV genome disclosed herein further comprises a transcription terminator (e.g., a polyadenylation sequence). In certain embodiments, the transcription terminator is 3' to the transgene. The transcription terminator may be any sequence that effectively terminates transcription, and one skilled in the art will understand that such a sequence may be isolated from any gene expressed in a cell in which transcription of at least a portion of an antibody coding sequence is desired. In certain embodiments, the transcription terminator comprises a polyadenylation sequence. In certain embodiments, the polyadenylation sequence is identical or substantially identical to the endogenous polyadenylation sequence of an immunoglobulin gene. In certain embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence. In certain embodiments, the polyadenylation sequence is an SV40 polyadenylation sequence (e.g., comprising a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or complementary to, SEQ ID NO:65, 68, or 69). In certain embodiments, the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO:65. In certain embodiments, the polyadenylation sequence consists of the nucleotide sequence set forth in SEQ ID NO:65. In certain embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence (e.g., comprising a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or a complementary nucleotide sequence to, SEQ ID NO:67). In certain embodiments, the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO:67. In certain embodiments, the polyadenylation sequence consists of the nucleotide sequence set forth in SEQ ID NO:67.

[0105] In certain embodiments, the rAAV genome comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in SEQ ID NO:71, 85, 86, 87, or 88. In certain embodiments, the editing element comprises a nucleotide sequence set forth in SEQ ID NO:71, 85, 86, 87, or 88. In certain embodiments, the editing element consists of a nucleotide sequence set forth in SEQ ID NO:71, 85, 86, 87, or 88.

[0106] In certain embodiments, the rAAV genome disclosed herein further comprises a 5' inverted terminal repeat (5'ITR) nucleotide sequence 5' of the TRE and a 3' inverted terminal repeat (3'ITR) nucleotide sequence 3' of the polyadenylation sequence associated with the antibody light chain coding sequence. ITR sequences from any AAV serotype or variants thereof may be used in the rAAV genome disclosed herein. The 5' and 3' ITRs may be from AAV of the same serotype or from AAV of different serotypes. Exemplary ITRs for use in the rAAV genome disclosed herein are set forth in SEQ ID NOs: 39, 40, 41, 42, 43 and 44 herein.

[0107] In certain embodiments, the 5'ITR or the 3'ITR is from AAV2. In certain embodiments, the 5'ITR and the 3'ITR are both from AAV2. In certain embodiments, the 5'ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:39, or the 3'ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:40. In certain embodiments, the 5' ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:39 and the 3' ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:40. In certain embodiments, the rAAV genome comprises a 5' ITR nucleotide sequence having the sequence of SEQ ID NO:39 and a 3' ITR nucleotide sequence having the sequence of SEQ ID NO:40.

[0108] In certain embodiments, the 5'ITR or the 3'ITR is from AAV5. In certain embodiments, the 5'ITR and the 3'ITR are both from AAV5. In certain embodiments, the 5' ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:42, or the 3' ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:43. In certain embodiments, the 5' ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:42 and the 3' ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:43. In certain embodiments, the rAAV genome comprises a 5' ITR nucleotide sequence having the sequence of SEQ ID NO:42 and a 3' ITR nucleotide sequence having the sequence of SEQ ID NO:43.

[0109] In certain embodiments, the 5' ITR nucleotide sequence and the 3' ITR nucleotide sequence are substantially complementary to each other (e.g., complementary to each other except for mismatches at 1, 2, 3, 4 or 5 nucleotide positions in the 5' or 3' ITR).

[0110] In certain embodiments, the 5' or 3' ITR is modified to reduce or eliminate degradation by Rep protein ("non-degradable ITR"). In certain embodiments, the non-degradable ITR comprises an insertion, deletion, or substitution in the nucleotide sequence of the final degradation site. Such modifications allow the formation of a self-complementary double-stranded DNA genome of AAV after the rAAV genome is replicated in an infected cell. Exemplary non-degradable ITR sequences are known in the art (see, for example, those set forth in U.S. Pat. Nos. 7,790,154 and 9,783,824, which are incorporated herein by reference in their entireties). In certain embodiments, the 5'ITR comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In certain embodiments, the 5'ITR consists of a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In certain embodiments, the 5'ITR consists of the nucleotide sequence set forth in SEQ ID NO: 41. In certain embodiments, the 3'ITR comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44. In certain embodiments, the 5'ITR consists of a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44. In certain embodiments, the 3'ITR consists of the nucleotide sequence set forth in SEQ ID NO:44. In a particular embodiment, the 5' ITR consists of the nucleotide sequence set forth in SEQ ID NO:41 and the 3' ITR consists of the nucleotide sequence set forth in SEQ ID NO:44.In a particular embodiment, the 5' ITR consists of the nucleotide sequence set forth in SEQ ID NO:41 and the 3' ITR consists of the nucleotide sequence set forth in SEQ ID NO:44.

[0111] In certain embodiments, 5'ITR is adjacent to an additional nucleotide sequence derived from wild-type AAV2 genome sequence.In certain embodiments, 5'ITR is adjacent to an additional 46bp sequence derived from wild-type AAV2 sequence adjacent to wild-type AAV2 ITR in AAV2 genome.In certain embodiments, the additional 46bp sequence is 3' to 5'ITR in rAAV genome.In certain embodiments, this 46bp sequence consists of the nucleotide sequence set forth in SEQ ID NO:45.

[0112] In certain embodiments, the 3'ITR is adjacent to an additional nucleotide sequence derived from the wild-type AAV2 genome sequence. In certain embodiments, the 3'ITR is adjacent to an additional 37 bp sequence derived from the wild-type AAV2 sequence adjacent to the wild-type AAV2 ITR in the AAV2 genome. See, for example, Savy et al., Human Gene Therapy Methods (2017) 28(5): 277-289, which is incorporated herein by reference in its entirety. In certain embodiments, the additional 37 bp sequence is 5' to the 3'ITR in the rAAV genome. In certain embodiments, this 37 bp sequence consists of the nucleotide sequence set forth in SEQ ID NO: 46.

[0113] In certain embodiments, the rAAV genome comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in SEQ ID NO: 75, 78, 80, 82, or 84. In certain embodiments, the editing element comprises a nucleotide sequence set forth in SEQ ID NO: 75, 78, 80, 82, or 84. In certain embodiments, the editing element consists of a nucleotide sequence set forth in SEQ ID NO: 75, 78, 80, 82, or 84.

[0114] AAV Rep protein The present disclosure provides a first nucleic acid vector comprising a Rep protein coding sequence or a coding sequence of a functional variant thereof. The expression of the AAV Rep gene is controlled by the use of two promoters and alternative splicing, resulting in four Rep proteins, Rep78, Rep68, Rep52 and Rep40. The Rep proteins are involved in AAV genome replication and viral genome packaging. The expression of the Rep proteins is controlled by the p5 and p19 promoters. The p5 promoter drives the expression of the alternative splice variants Rep78 and Rep68. The p19 promoter drives the expression of the alternative splice variants Rep52 and Rep40. Thus, the first nucleic acid vector may comprise a nucleotide sequence encoding one or more Rep proteins or functional variants thereof.

[0115] One or more Rep proteins can be derived from AAV2. An exemplary AAV2 genome sequence can be found through NCBI reference sequence NC_001401.2. According to the NCBI reference sequence, Rep68 is encoded by nucleotides 321-2252; Rep78 is encoded by nucleotides 321-2186; Rep40 is encoded by nucleotides 993-2252; and Rep52 is encoded by nucleotides 993-2186.

[0116] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding Rep78, wherein the nucleotide sequence encoding Rep78 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 50. In certain embodiments, the nucleotide sequence encoding Rep78 comprises or consists of the sequence set forth in SEQ ID NO: 50. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep78 comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep78. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep78 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 47. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep78 comprises or consists of the sequence set forth in SEQ ID NO: 47. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep78 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 51. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep78 comprises or consists of the sequence set forth in SEQ ID NO: 51. In certain embodiments, the disclosure provides nucleic acids comprising nucleotide sequences corresponding to sequences encoding Rep78 described for AAV2 in different adenovirus serotypes.

[0117] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding Rep68, wherein the nucleotide sequence encoding Rep68 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 52. In certain embodiments, the nucleotide sequence encoding Rep68 comprises or consists of the sequence set forth in SEQ ID NO: 52. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep68 comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep68. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep68 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 47. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep68 comprises or consists of the sequence set forth in SEQ ID NO: 47. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep68 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 53. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep68 comprises or consists of the sequence set forth in SEQ ID NO: 53. In certain embodiments, the present disclosure provides nucleic acids comprising nucleotide sequences corresponding to sequences encoding Rep68 described for AAV2 in different adenovirus serotypes.

[0118] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding Rep40, wherein the nucleotide sequence encoding Rep40 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 54. In certain embodiments, the nucleotide sequence encoding Rep40 comprises or consists of the sequence set forth in SEQ ID NO: 54. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep40 comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep40. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep40 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 48. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep40 comprises or consists of the sequence set forth in SEQ ID NO: 48. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep40 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 55. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep40 comprises or consists of the sequence set forth in SEQ ID NO: 55. In certain embodiments, the disclosure provides nucleic acids comprising nucleotide sequences corresponding to sequences encoding Rep40 described for AAV2 in different adenovirus serotypes.

[0119] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding Rep52, wherein the nucleotide sequence encoding Rep52 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 56. In certain embodiments, the nucleotide sequence encoding Rep52 comprises or consists of the sequence set forth in SEQ ID NO: 56. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep52 comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep52. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep52 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 48. In certain embodiments, the transcriptional regulatory element operably linked to the nucleotide sequence encoding Rep52 comprises or consists of the sequence set forth in SEQ ID NO: 48. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep52 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 57. In certain embodiments, the nucleic acid comprising the nucleotide sequence encoding Rep52 comprises or consists of the sequence set forth in SEQ ID NO: 57. In certain embodiments, the disclosure provides nucleic acids comprising nucleotide sequences corresponding to sequences encoding Rep52 described for AAV2 in different adenovirus serotypes.

[0120] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding Rep78, Rep68, Rep40 and Rep52, wherein the nucleotide sequence encoding Rep78, Rep68, Rep40 and Rep52 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 58. In certain embodiments, the nucleotide sequence encoding Rep78, Rep68, Rep40 and Rep52 comprises or consists of the sequence set forth in SEQ ID NO: 58. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep78, Rep68, Rep40 and Rep52 comprises one or more transcriptional regulatory elements that can be operably linked to each of the nucleotide sequences encoding Rep78, Rep68, Rep40 and Rep52. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep78, Rep68, Rep40 and Rep52 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 59. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding Rep78, Rep68, Rep40 and Rep52 comprises or consists of the sequence set forth in SEQ ID NO:59.

[0121] AAV Capsid Protein The present disclosure provides a first nucleic acid vector comprising a nucleotide sequence comprising an AAV capsid protein coding sequence. The first nucleic acid vector may comprise a nucleotide sequence encoding an AAV capsid protein from any AAV capsid known in the art, including naturally occurring AAV isolates and variants thereof.

[0122] AAV capsid proteins include VP1, VP2, and VP3 capsid proteins. VP1, VP2, and / or VP3 capsid proteins assemble into a capsid that surrounds the rAAV genome. In certain embodiments, assembly of the capsid proteins is promoted by assembly activating proteins (AAPs). The capsids of certain AAV serotypes require the role of AAPs in transporting the capsid proteins to the nucleolus for assembly. For example, AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV12 require AAPs to form capsids, while AAV4, AAV5, and AAV11 capsids can assemble without AAPs. See, e.g., Earley et al. (2017) J. Virol. 91(3):e01980-16.

[0123] Different AAV serotypes or variants thereof contain AAV capsid proteins with different amino acid sequences. Suitable AAV capsid proteins include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, AAVr The capsid protein includes, but is not limited to, capsid proteins from AAVhsC1, AAVhsC2, AAVhsC3, AAVhsC4, AAVhsC5, AAVhsC6, AAVhsC7, AAVhsC8, AAVhsC9, AAVhsC10, AAVhsC11, AAVhsC12, AAVhsC13, AAVhsC14, AAVhsC15, AAVhsC16, AAVhsC17, and any variants thereof. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhlO, and AAVrh74. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, and AAVrh74. The sequences of various AAV capsid proteins are disclosed, for example, in U.S. Patent Publication Nos. US20030138772, US20140359799, US20150159173, US20150376607, US20170081680, and US20170360962A1, and PCT Publication No. WO2020227515, which are incorporated by reference in their entireties.

[0124] For example, in certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.In certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17, and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G; and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the capsid protein comprises the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0125] For example, in certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 138 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.In certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17, and an amino acid sequence in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16. the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO:16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In a specific embodiment, the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G.In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the capsid protein comprises the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0126] For example, in certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 1 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17. In certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17;the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO:16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO:16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO:16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO:16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO:16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T;and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the capsid protein is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15,Contains 16 or 17 amino acids 1 to 736 of the amino acid sequence.

[0127] In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203 to 736 of SEQ ID NO:1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17; (b) a capsid protein having an amino acid sequence consisting of amino acids 138 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1 to 736 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

[0128] In certain embodiments, the AAV capsid comprises: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 203 to 736 of SEQ ID NO:8; or (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 100% sequence identity to the sequence of amino acids 138 to 736 of SEQ ID NO:8. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:8; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:8. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 8; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 8; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 8. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 8; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 8; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 8. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203 to 736 of SEQ ID NO:8; (b) a capsid protein having an amino acid sequence consisting of amino acids 138 to 736 of SEQ ID NO:8; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1 to 736 of SEQ ID NO:8.

[0129] In certain embodiments, the AAV capsid comprises: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 203 to 736 of SEQ ID NO:11; or (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 100% sequence identity to the sequence of amino acids 138 to 736 of SEQ ID NO:11. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:11; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:11. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 11; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 11; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 11. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 11; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 11; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 11. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203 to 736 of SEQ ID NO:11; (b) a capsid protein having an amino acid sequence consisting of amino acids 138 to 736 of SEQ ID NO:11; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1 to 736 of SEQ ID NO:11.

[0130] In certain embodiments, the AAV capsid comprises: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 203 to 736 of SEQ ID NO:13; or (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 100% sequence identity to the sequence of amino acids 138 to 736 of SEQ ID NO:13. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:13; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:13. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 13; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 13; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 13. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 13; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 13; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 13. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203 to 736 of SEQ ID NO: 13; (b) a capsid protein having an amino acid sequence consisting of amino acids 138 to 736 of SEQ ID NO: 13; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1 to 736 of SEQ ID NO: 13.

[0131] In certain embodiments, the AAV capsid comprises: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids 203 to 736 of SEQ ID NO:16; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids 138 to 736 of SEQ ID NO:16; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids 1 to 736 of SEQ ID NO:16. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 16; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 16; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 16. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 16; (b) a capsid protein comprising the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 16; and (c) a capsid protein comprising the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 16. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203 to 736 of SEQ ID NO:16; (b) a capsid protein having an amino acid sequence consisting of amino acids 138 to 736 of SEQ ID NO:16; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1 to 736 of SEQ ID NO:16.

[0132] In certain embodiments, the nucleotides encoding the AAV capsid protein are operably linked to a transcriptional regulatory element that controls the expression of the AAV capsid protein. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a natural promoter. Any promoter known in the art that can control the expression of the AAV capsid protein may be used. Promoters suitable for use are known to those skilled in the art and include, but are not limited to, p40 promoter, metallothionine (MT) promoter, mouse mammary tumor virus (MMTV) promoter, T7 promoter, ecdysone insect promoter, tetracycline-repressible promoter, tetracycline-inducible promoter, RU486-inducible promoter, and rapamycin-inducible promoter. Other suitable promoters include, but are not limited to, CMV promoter, CBA promoter, and CAG promoter.

[0133] In certain embodiments, the transcriptional regulatory element operably linked to a nucleotide sequence encoding an AAV capsid protein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 47, 48 or 49. In certain embodiments, the transcriptional regulatory element operably linked to a nucleotide sequence encoding an AAV capsid protein comprises or consists of the sequence set forth in SEQ ID NO: 47, 48 or 49.

[0134] In another aspect, the present disclosure provides a first nucleic acid vector comprising a first nucleotide sequence comprising a Rep-Cap element and a second nucleotide sequence comprising a rAAV genome comprising a transgene. In certain embodiments, the Rep-Cap element comprises a nucleic acid sequence encoding an AAV Rep protein and a nucleic acid sequence encoding an AAV capsid protein. The Rep-Cap element may comprise a nucleic acid sequence encoding any AAV Rep protein known in the art and a nucleic acid sequence encoding any AAV capsid protein known in the art. In certain embodiments, the Rep-Cap element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: 73 or 77.

[0135] III. Second Nucleic Acid Vector The dual vector transfection system described herein generally includes a second nucleic acid vector that includes one or more helper virus genes. As will be understood by those skilled in the art, AAV replication depends on the presence of helper factors encoded by helper virus genes. Helper factors can be provided through co-infection with helper viruses, such as, but not limited to, helper viruses from adenovirus, herpesvirus, papillomavirus, cytomegalovirus, baculovirus and human bocavirus. However, growing AAV in the presence of helper virus can lead to host cell lysis and / or AAV product contamination. Therefore, helper virus genes encoding the helper factors required for AAV replication can be provided in the vector used to transfect host cells.

[0136] The dual vector transfection system described herein generally involves transfecting a host cell for AAV (e.g., rAAV) production with two nucleic acid vectors: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising helper virus genes. In certain embodiments, the second nucleic acid vector does not comprise any of the components of AAV production found in the first nucleic acid vector. In certain embodiments, the second nucleic acid vector does not comprise a rAAV genome comprising a transgene. In certain embodiments, the second nucleic acid vector does not comprise an AAV capsid protein coding sequence. In certain embodiments, the second nucleic acid vector does not comprise a coding sequence for a Rep coding sequence or a functional fragment thereof. In certain embodiments, the second nucleic acid vector does not comprise a rAAV genome including a transgene, the second nucleic acid vector does not comprise an AAV capsid protein coding sequence, and / or the second nucleic acid vector does not comprise a Rep coding sequence or a coding sequence for a functional fragment thereof.

[0137] In certain embodiments, the second nucleic acid vector comprises at least one helper virus gene, which may be derived from a helper virus selected from the group consisting of adenovirus, herpesvirus, poxvirus, cytomegalovirus, and baculovirus. The helper virus gene may be operably linked to a transcriptional regulatory element that controls expression of the helper virus gene. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a native promoter. Promoters suitable for use are known to those skilled in the art and include, but are not limited to, RSV LTR promoter, CMV immediate early promoter, SV40 promoter, dihydrofolate reductase promoter, cytoplasmic β-actin promoter, phosphoglycerate kinase (PGK) promoter, metallothionine (MT) promoter, mouse mammary tumor virus (MMTV) promoter, T7 promoter, ecdysone insect promoter, tetracycline-repressed promoter, tetracycline-inducible promoter, RU486-inducible promoter, and rapamycin-inducible promoter.

[0138] In certain embodiments, the second nucleic acid vector comprises at least one helper virus gene. The at least one helper virus gene may be derived from adenovirus (AdV). The minimal set of AdV helper factors known to be necessary for efficient AAV production consists of AdV molecules El, E2, E4 and VA RNA (see, e.g., Meier et al. (2020) Virus 12(6):662). In particular, the minimal set of AdV helper factors necessary for efficient AAV production includes AdV molecules E1A, E1B, E2A, E4 and VA RNA. In certain embodiments, the second nucleic acid vector comprises a sufficient set of helper virus genes to allow efficient AAV production (e.g., AAV replication and packaging) in a host cell (e.g., a host AAV-producing cell).

[0139] A typical AdV genome expresses about 40 tightly regulated proteins, divided into early and late phases. The early phase proteins include E1A, E1B, E2A, and E4. Briefly, E1A and E2A proteins function to activate the AAV promoters p5 and p19, which control the expression of the AAV Rep protein. E1A-mediated p5 activity has been shown to be required for AAV replication. E2A is a single-stranded DNA binding protein that has been shown to promote various aspects of AAV replication. The E1B gene encodes the E1B19K and E1B55K oncoproteins. E1B19K inhibits E1A-induced apoptosis, and E1B55K inhibits the tumor suppressor protein p53. E1B55K functions together with E4orf6 to promote AAV second strand synthesis and viral DNA replication. E1B55K has also been shown to facilitate AAV mRNA export and promote AAV gene expression while inhibiting cellular mRNA export. E1B19K was found to function in enhancing AAV titers when co-expressed with other AdV helper factors such as E1A, E1B55K, E2A, and E4orf6.

[0140] VA RNA has been found to function in the inhibition of the cellular innate immune protein double-stranded RNA-activated kinase (PKR), and inhibition of this kinase ensures efficient viral protein synthesis. VA RNA has also been shown to facilitate the synthesis and assembly of AAV structural proteins. It is readily understood by those skilled in the art that the VA nucleic acid in the AdV genome is a non-translated nucleic acid sequence that gives rise to VA RNA.

[0141] One of the most commonly used helper functions is derived from human AdV5 type. Adenovirus helper virus genes may also be derived from other known adenoviruses, such as AdV2 type. The AdV5 genome is approximately 36 kilobases, and an exemplary AdV5 genome sequence can be found via the NCBI reference sequence AC_000008.1. According to the NCBI reference sequence, E1A is encoded by nucleotides 560-1545; E1B19K is encoded by nucleotides 1714-2244; E1B55K is encoded by nucleotides 2019-3509; E2A is encoded by nucleotides 22443-24032; and E4orf6 / 7 is encoded by nucleotides 32914-34077.

[0142] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding AdV5 E2A. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E2A comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding AdV5 E2A. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E2A comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:60. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E2A comprises or consists of the sequence set forth in SEQ ID NO:60. In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence corresponding to the sequence encoding E2A described for AdV5 in a different adenovirus serotype (e.g., AdV2).

[0143] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding AdV5 E4. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E4 comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding AdV5 E4. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E4 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:61. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E4 comprises or consists of the sequence set forth in SEQ ID NO:61. In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence corresponding to the sequence encoding E4 described for AdV5 in a different adenovirus serotype (e.g., AdV2).

[0144] In certain embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an AdV5 VA RNA. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding an AdV5 VA RNA comprises a transcriptional regulatory element operably linked to the nucleotide sequence encoding the AdV5 VA RNA. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding an AdV5 VA RNA comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:62. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding an AdV5 VA RNA comprises or consists of the sequence set forth in SEQ ID NO:62. It is readily understood by those skilled in the art that the nucleic acid sequence of a VA RNA is a non-translated nucleic acid sequence that gives rise to (e.g., "encodes") a VA RNA. In certain embodiments, the disclosure provides nucleic acids comprising a nucleotide sequence in a different adenovirus serotype (eg, AdV2) that corresponds to a sequence encoding a VA RNA described for AdV5.

[0145] In certain embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding AdV5 E2A, E4 and VA RNA. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E2A, E4 and VA RNA comprises one or more transcriptional regulatory elements that can be operably linked to each of the nucleotide sequences encoding AdV5 E2A, E4 and VA RNA. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E2A, E4 and VA RNA comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:63. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding AdV5 E2A, E4 and VA RNA comprises or consists of the sequence set forth in SEQ ID NO:63.

[0146] In certain embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a minimal set of AdV helper factors required for efficient AAV production, hi certain embodiments, the nucleic acid comprising nucleotides encoding the minimal set of AdV helper factors encodes the AdV molecules E1A, E1B, E2A, E4 and VA RNA.

[0147] Certain host cells, such as HEK293T cells, may endogenously provide some, but not all, of the necessary helper factors, and exogenously provide the remaining helper factors by plasmid transfection. For example, HEK293T cells endogenously express adenovirus E1A and E1B genes and provide the remaining adenovirus helper genes, i.e., those encoding AdV5 E4, E2A, and virus-associated (VA) RNA. Such AdV5 helper genes may be provided by a single vector through transfection. In certain embodiments, the present disclosure provides a second nucleic acid vector comprising AdV5 helper virus genes selected from the group consisting of E2A, E4, and VA RNA. In certain embodiments, the present disclosure provides a second nucleic acid vector comprising helper virus genes encoding E2A, E4, and VA RNA as described for AdV5 from a different adenovirus serotype (e.g., AdV2).

[0148] Helper virus genes may also be derived from herpesviruses, papillomaviruses, and human bocaviruses. Examples of herpesviruses from which helper virus factors can be derived include HSV-1 and HSV-2. Helper virus factors from HSV-1 known to be involved in supporting AAV production include, but are not limited to, UL5, UL8, UL52, ICP8, ICP0, ICP4, ICP22, UL30, and UL42. The various functions of these HSV-1 helper virus factors and the way in which they support AAV production are known to those skilled in the art. For example, in addition to the single-stranded DNA binding protein ICP8, the HSV-1 helicase-primase complex UL5 / UL8 / UL52 is also known to be sufficient to restore AAV progeny production in AAV infection models; ICP0, ICP4, and ICP22 are involved in promoting the expression of Rep proteins; and the HSV-1 DNA polymerase UL30 / UL42 is involved in AAV DNA replication. Thus, in certain embodiments, the second nucleic acid vector comprises at least one helper virus gene selected from the group consisting of UL5, UL8, UL52, ICP8, ICP0, ICP4, ICP22, UL30 and UL42. An example of a papillomavirus from which the helper virus factor can be derived is HPV-16. In certain embodiments, the helper virus factor from HPV-16 can enhance AAV production in the presence of AdV helper factors. Such HPV-16 helper factors known to be involved in supporting AAV replication include, but are not limited to, E1, E2 and E6. An example of a human bocavirus from which the helper virus factor can be derived is human bocavirus 1 (HBoV1). Helper virus factors from HBoV1 known to be involved in supporting AAV production include, but are not limited to, NP1, NS2, NS4 and the viral long non-coding RNA BocaSR.

[0149] IV. Vectors and Cells The disclosure provides a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising an rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and a second nucleic acid vector comprising helper virus genes.

[0150] The first and second nucleic acid vectors may be, independently, any form of nucleic acid vector. Suitable vectors include, but are not limited to, plasmids, minimal vectors (e.g., minicircles, Nanoplasmids™, doggybone, MIDGE vectors, etc.), viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. In certain embodiments, the first and / or second nucleic acid vectors are DNA plasmids or DNA minimal vectors. Any DNA plasmid or DNA minimal vector that can accommodate the required vector elements may be used for the first and second nucleic acid vectors. Suitable DNA minimal vectors include, but are not limited to, linear covalently closed DNA (e.g., ministring DNA), linear covalently closed dumbbell-shaped DNA (e.g., doggybone DNA, dumbbell DNA), minicircles, Nanoplasmids™, minimalistic immunologically defined gene expression (MIDGE) vectors, and others known to those skilled in the art. DNA minimal vectors and methods for their production are described, for example, in U.S. Patent Application Nos. 20100233814, 20120282283, 20130216562, 20150218565, 20150218586, 20160008488, 20160215296, 20160355827, 20190185924, 20200277624, and 20210010021, all of which are incorporated by reference in their entireties.

[0151] In certain embodiments, the nucleic acid in the vector disclosed herein is optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and / or removal of mRNA instability elements. Methods for producing polynucleotides optimized for recombinant expression by introducing codon changes and / or removing inhibitory regions in the mRNA can be performed, for example, by adapting the optimization methods described in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) in the RNA can be mutated without changing the amino acid encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These modifications take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In certain embodiments, conservative mutations, e.g., altering one or more codons to code for similar amino acids with similar chemical structures and properties and / or function as the original amino acid, may be desirable. Such methods may increase expression of the encoded capsid protein compared to expression of a capsid encoded by a non-optimized polynucleotide.

[0152] The vectors disclosed herein can be introduced into cells (using any technique known in the art) for propagation of the vector and / or expression of the protein encoded by the vector. Thus, in another aspect, the present disclosure provides a recombinant cell comprising the vector disclosed herein. Additionally, in another aspect, the present disclosure provides a method for producing rAAV, the method comprising culturing a recombinant cell under conditions in which the polynucleotide is expressed and rAAV is produced.

[0153] A variety of host cells and expression systems may be utilized. Such expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also represent cells that may produce rAAV when transformed or transfected with the appropriate nucleotide coding sequences described herein. These include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli and B. subtilis) transformed with, for example, a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector comprising the nucleotide coding sequences described herein; yeast (e.g., Saccharomyces Pichia) transformed with, for example, a recombinant yeast expression vector comprising the nucleotide coding sequences described herein; insect cell systems infected with, for example, a recombinant virus expression vector (e.g., baculovirus) comprising the nucleotide coding sequences described herein; plant cell systems (e.g., Chlamydomonas spp.) infected with, for example, a recombinant virus expression vector (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with, for example, a recombinant plasmid expression vector (e.g., Ti plasmid) comprising the nucleotide coding sequences described herein. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK293T, HEK293F, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct that includes a nucleotide coding sequence described herein, including, for example, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a vaccinia virus 7.5K promoter). In certain embodiments, the cell expressing the nucleotide coding sequence described herein is a human cell, e.g., a human cell line. In certain embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3.In certain embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used to express the nucleotide coding sequences described herein. For example, mammalian cells, such as CHO or HEK293 cells, in combination with vector elements, such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for the polynucleotides described herein.

[0154] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use for the expressed protein. For example, when producing large amounts of protein, a vector that directs the expression of a high level of fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), in which protein coding sequences can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509); and the like, all of which are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from solubilized cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain cleavage sites for thrombin or factor Xa protease so that the cloned target gene product can be released from the GST moiety.

[0155] In an insect system, for example, Autographa cahfornica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The protein coding sequence may be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0156] In mammalian host cells, many virus-based expression systems can be utilized. When adenovirus is used as an expression vector, the protein coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions El or E3) produces recombinant viruses that are viable and capable of expressing the nucleotide coding sequences described herein in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9, which is incorporated herein by reference in its entirety). Specific initiation signals can also be required for efficient translation of the inserted protein coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, to ensure translation of the entire insert, the initiation codon must be in phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter Get al. (1987) Methods Enzymol. 153:516-544, the entire contents of which are incorporated herein by reference).

[0157] In addition, a host cell strain may be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system may be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, HEK293T, HEK293F, HEK293EBNA, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CAP, CAP-T, CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, AGE1.CR, A549, HsS78Bst, HepG2, C139, EB66, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0158] In certain embodiments, rather than using an expression vector containing a viral origin of replication, a host cell may be transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and a selection marker. Following introduction of the polynucleotide, the engineered cells may be grown in rich medium for 1-2 days and then switched to selective medium. The selection marker in the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method may be advantageously used to engineer cell lines that express the proteins or fragments thereof described herein.

[0159] A number of selection systems may be used in tk, hgprt or aprt cells, respectively, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3):817-23) genes, all of which are incorporated by reference herein in their entireties. Resistance to antimetabolites may also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al. (1980) PNAS 77(6):3567-70; O'Hare K et al. (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al. (1984) Gene 30(1-3):147-56) (all of which are incorporated herein by reference in their entireties).Methods generally known in the art of recombinant DNA technology are routinely applied to select the desired recombinant clones, such methods being described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al. (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entirety.

[0160] V. Adeno-Associated Virus Packaging Systems and Methods In another aspect, the present disclosure provides a packaging system for the recombinant preparation of recombinant adeno-associated virus (rAAV) disclosed herein. In particular, the present disclosure provides a packaging system useful for AAV production under the dual vector transfection system described herein (e.g., AAV production is mediated by the use of a packaging system comprising a first and a second nucleic acid vector delivered to a host cell). Such a packaging system generally comprises or consists of: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene. The first and second nucleic acid vectors together can provide all the components necessary for the production of rAAV. In certain embodiments, the components necessary for the manufacture of rAAV are provided by the host cell in which rAAV is produced. In such embodiments, the first and second nucleic acid vectors, together with the host cell, can provide all the components necessary for the production of rAAV, and the packaging system described herein operates within the cell to encapsidate the rAAV genome to form the rAAV.

[0161] In certain embodiments, the present disclosure provides an rAAV packaging system comprising: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising helper virus genes. In certain embodiments, the present disclosure provides an rAAV packaging system comprising: (1) a first nucleic acid vector comprising, in a 5' to 3' direction, a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising helper virus genes.

[0162] In certain embodiments, the first nucleic acid vector of the packaging system comprises a rAAV genome comprising a transgene. The first nucleic acid vector of the packaging system of the present disclosure further comprises a coding sequence for an AAV Rep protein or a functional variant thereof, and an AAV capsid protein coding sequence. Thus, the present disclosure provides a first nucleic acid vector of the packaging system comprising a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein. In certain embodiments, the first nucleic acid vector of the packaging system comprises, in the 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof, a second nucleotide sequence comprising a rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein. In certain embodiments, the first nucleic acid vector of the packaging system does not comprise a helper virus gene.

[0163] Any AAV Rep protein may be used in the packaging system disclosed herein. In certain embodiments of the packaging system, the Rep nucleotide sequence encodes an AAV2 Rep protein. Suitable AAV2 Rep proteins may include, but are not limited to, Rep78 / 68 or Rep68 / 52. In certain embodiments of the packaging system, the nucleotide sequence encoding the AAV2 Rep protein comprises a nucleotide sequence encoding a protein having a minimum percent sequence identity to the AAV2 Rep amino acid sequence of SEQ ID NO:64, the minimum percent sequence identity being at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) over the length of the amino acid sequence of the AAV2 Rep protein. In certain embodiments of the packaging system, the AAV2 Rep protein has the amino acid sequence set forth in SEQ ID NO:64.

[0164] In certain embodiments, the second nucleic acid vector of the packaging system comprises a helper virus gene. The second nucleic acid vector of the packaging system of the present disclosure may comprise one or more helper virus genes. Certain aspects of the present disclosure indicate that the second nucleic acid vector of the packaging system does not comprise any components of AAV production found in the first nucleic acid vector described herein. In certain embodiments, the second nucleic acid vector of the packaging system does not comprise a rAAV genome including a transgene. In certain embodiments, the second nucleic acid vector of the packaging system does not comprise an AAV capsid protein coding sequence. In certain embodiments, the second nucleic acid vector of the packaging system does not comprise a Rep coding sequence or a coding sequence of a functional variant thereof. In certain embodiments, the second nucleic acid vector of the packaging system does not comprise a rAAV genome including a transgene, the second nucleic acid vector of the packaging system does not comprise an AAV capsid protein coding sequence, and / or the second nucleic acid vector of the packaging system does not comprise a Rep coding sequence or a coding sequence of a functional variant thereof.

[0165] In certain embodiments of the packaging system, the helper virus is selected from the group consisting of adenovirus, herpes virus (e.g., herpes simplex virus (HSV), pox virus (such as vaccinia virus), cytomegalovirus (CMV), and baculovirus. In certain embodiments of the packaging system where the helper virus is adenovirus, the adenovirus genome comprises one or more adenovirus RNA genes selected from the group consisting of El, E2, E4, and VA. In certain embodiments of the packaging system where the helper virus is HSV, the HSV genome comprises one or more HSV genes selected from the group consisting of UL5 / 8 / 52, ICP0, ICP4, ICP22, and UL30 / UL42.

[0166] In certain embodiments of the packaging system, the first and second nucleic acid vectors of the packaging system are contained within two plasmids. In certain embodiments, the first nucleic acid vector of the packaging system is contained within a first plasmid. In certain embodiments, the second nucleic acid vector of the packaging system is contained within a second plasmid.

[0167] In certain embodiments of the packaging system, the first and second nucleic acid vectors of the packaging system are contained within two recombinant helper viruses. In certain embodiments, the first nucleic acid vector of the packaging system is contained within a first recombinant helper virus. In certain embodiments, the second nucleic acid vector of the packaging system is contained within a second recombinant helper virus. In certain embodiments, the first and second nucleic acid vectors of the packaging system are contained within a single recombinant helper virus.

[0168] In a further aspect, the present disclosure provides a method for recombinant preparation of rAAV, comprising transfecting or transducing a cell with a packaging system described herein under conditions operable to encapsidate the rAAV genome to form an rAAV. Exemplary methods for recombinant preparation of rAAV include transient transfection (e.g., with one or more transfection plasmids), viral infection (e.g., with one or more recombinant helper viruses, such as adenovirus, poxvirus (e.g., vaccinia virus), herpes virus (e.g., HSV, cytomegalovirus, or baculovirus), and stable producer cell line transfection or infection (e.g., with stable producer cells, such as mammalian or insect cells).

[0169] Thus, the present disclosure provides a packaging system for the preparation of rAAV, the packaging system comprising: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof; a second nucleotide sequence comprising an rAAV genome; and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene. In certain embodiments, the present disclosure provides a packaging system for the preparation of rAAV, the packaging system comprising: (1) a first nucleic acid vector comprising, in a 5' to 3' direction, a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof; a second nucleotide sequence comprising an rAAV genome; and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene.

[0170] Thus, the present disclosure provides a method for the recombinant preparation of rAAV, comprising transfecting or transducing a cell with a packaging system comprising: (1) a first nucleic acid vector comprising: a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof; a second nucleotide sequence comprising an rAAV genome; and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene. In certain embodiments, the present disclosure provides a method for the recombinant preparation of rAAV, comprising transfecting or transducing a cell with a packaging system comprising: (1) a first nucleic acid vector comprising: in a 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof; a second nucleotide sequence comprising an rAAV genome; and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene.

[0171] In certain embodiments, the total amount of nucleic acid transfected or transduced into a cell includes (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein or a functional variant thereof; a second nucleotide sequence comprising an rAAV genome; and a third nucleotide sequence encoding an AAV capsid protein, and (2) a second nucleic acid vector comprising helper virus genes, which is between 0.1 μg DNA / 1E6 cell and 4 μg DNA / 1E6 cell. For example, the total amount of nucleic acid transfected or transduced into the cell, including the first and second nucleic acid vectors, is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 μg of DNA / 1E6 cells. In certain embodiments, the total amount of nucleic acid transfected or transduced into the cell, including the first and second nucleic acid vectors, is 1 μg of DNA / 1E6 cells. In certain embodiments, the total amount of nucleic acid transfected or transduced into the cell, comprising the first and second nucleic acid vectors, is 0.6 μg DNA / 1E6 cells. In certain embodiments, the total amount of nucleic acid transfected or transduced into the cell, comprising the first and second nucleic acid vectors, is 0.7 μg DNA / 1E6 cells. In certain embodiments, the total amount of nucleic acid transfected or transduced into the cell, comprising the first and second nucleic acid vectors, is 0.75 μg DNA / 1E6 cells. In certain embodiments, the total amount of nucleic acid transfected or transduced into the cell, comprising the first and second nucleic acid vectors, is 0.8 μg DNA / 1E6 cells. In certain embodiments, the total amount of nucleic acid transfected or transduced into the cell, comprising the first and second nucleic acid vectors, is 0.9 μg DNA / 1E6 cells.

[0172] In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is from 1:0.1 to 1:20. For example, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3 .2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, or 1:20. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is selected from the group consisting of 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:3, or 1:4. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:2. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.2 to 1:1. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.6. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.8. In certain embodiments, the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:1.

[0173] In certain embodiments, the methods of recombinant preparation of rAAV disclosed herein result in increased rAAV titers compared to methods comprising producing rAAV using a mammalian cell comprising: (i) a first vector comprising a nucleotide sequence encoding an AAV Rep protein and an AAV capsid protein; (ii) a second vector comprising an rAAV genome; and (iii) a third vector comprising one or more helper virus genes. In certain embodiments, the methods of recombinant preparation of rAAV disclosed herein result in increased rAAV titers compared to methods comprising producing rAAV using a mammalian cell comprising: (i) a first vector comprising a nucleotide sequence encoding an AAV Rep protein and an AAV capsid protein; (ii) a second vector comprising an rAAV genome; and (iii) a third vector comprising one or more helper virus genes.

[0174] In certain embodiments, the mammalian cells are provided in a cell culture. In certain embodiments, the cell culture has a volume of at least 2 liters, at least 50 liters, or at least 2000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 5000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 4000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 3000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 2500 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 2000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 1500 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 1000 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 500 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 250 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 100 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 50 liters. In certain embodiments, the cell culture has a volume of about 2 liters to about 25 liters. In certain embodiments, the methods described herein are performed in a bioreactor having a volume of at least 2 liters, at least 50 liters, or at least 2000 liters. In certain embodiments, the methods described herein are performed in a bioreactor having a volume of 2000 liters. EXAMPLES

[0175] The following examples are offered by way of illustration and not by way of limitation.

[0176] Example 1: Materials and Methods The following general materials and methods were used in the examples below.

[0177] Small scale production: HEK293 cells were grown for at least one passage and inoculated into shake flasks containing the appropriate amount of cell culture medium prior to transfection. The shake flasks were incubated in a shaker at 37°C, 8% CO2, and 135 rpm. When the cells reached a density of 1.8E6-2.4E6 cells / mL (for Examples 1-8) or 3.6E6-5E6 cells / mL (for Example 9), the cells were transfected. The transfection mix was prepared by mixing calculated volumes of vector(s), OptiPro medium, and polyethyleneimine (PEI), all at ambient temperature. The transfection mix was then added to the shake flasks and incubated in a shaker at 37°C, 8% CO2, and 135 rpm for 72 hours before harvesting. After 72 hours of incubation, cells were lysed using lysis buffer containing 1M Tris (pH 9.5), 10% Triton X-100, 1M MgCl2, endonuclease (e.g., BENZONASE®, DENARASE®) and 5M NaCl, and shake flasks were incubated at 37°C, 8% CO2, and 135 rpm for 60 minutes. Crude lysate samples were collected by centrifugation.

[0178] Preparation of 2L bioreactor: HEK293 cells were grown for at least one passage and inoculated into a 2L bioreactor (Millipore Mobius) containing the appropriate amount of cell culture medium prior to transfection. The pH was changed to 7.1 ± 0.1 pre-transfection and cells were transfected at a density of 1.8E6-2.4E6 cells / mL (for Examples 4-8) or 3.6E6-5E6 cells / mL (for Examples 9-11). Transfection mix was prepared by mixing calculated volumes of vector(s), OptiPro SFM media, and polyethylenimine (PEI), all at ambient temperature, and allowed to equilibrate for 10 minutes before adding the transfection mix to the cells. Cells were harvested 69-75 hours post-transfection. Harvested cells were lysed using a lysis buffer containing 1 M Tris (pH 9.5), 10% Triton X-100, 1 M MgCl2, an endonuclease (e.g., BENZONASE®, DENARASE®) and 5 M NaCl. An appropriate volume of lysis buffer was added to the bioreactor and cells were incubated at 37° C., 283 rpm for 120 minutes. Crude lysate samples were collected after centrifugation to remove cell debris.

[0179] Vector genome productivity in vector genomes per cell (vg / cell) was determined by droplet digital PCR (ddPCR) by standard methods using primer / probe sets specific for the transgene payload of the transgene-containing vector (i.e., the transgene vector). Vector genome productivity in vector genomes per liter (vg / L) was determined by droplet digital PCR (ddPCR) by standard methods using primer / probe sets specific for the transgene payload of the transgene-containing vector (i.e., the transgene vector). The number of capsids per cell was determined using enzyme-linked immunosorbent assay (ELISA) by standard methods using immobilized antibodies directed against an epitope of the capsid encoded by the vector containing the Cap sequence. The percentage of intact vector genomes (i.e., the percentage of complete capsids) was calculated by dividing the vector genome productivity measured by ddPCR by the number of capsids per cell measured by ELISA (Examples 2-4) or determined by analytical ultracentrifugation sedimentation velocity (AUC) analysis (Example 5).

[0180] Example 2: Comparison between dual and triple transfection systems A first small-scale manufacturing proof-of-concept study was performed to evaluate the utility of the dual vector transfection system in terms of its vector genome (VG) productivity and percentage of intact vector genome that could be obtained in comparison with the triple transfection system. The transfection conditions were set as described in Table 1. [Table 1]

[0181] The dual vector transfection system contained a first V4 vector and a second V3 vector, as described in Table 1. The triple vector transfection system contains vectors V1, V2 and V3. In Table 1, the vector ratios are based on mass. The elements contained in the various vectors are described in Table 2.

[0182] Transfection mixtures for each transfection condition were prepared in appropriately sized conical tubes by adding calculated volumes of vector(s), OptiPro medium, and polyethyleneimine (PEI), all at ambient temperature. Transfection mixtures were added to cells at a concentration of 1 μg DNA / 1E6 cells. Shake flasks were incubated for 72 hours before harvesting. At harvest, cells were lysed and crude lysate samples were collected after centrifugation to remove cell debris for subsequent droplet digital PCR (ddPCR) and capsid analysis by ELISA. [Table 2-1] [Table 2-2]

[0183] Figures 1A-1C show the VG productivity (Figure 1A), capsid productivity (Figure 1B), and percentage of intact vector genome (Figure 1C) obtained from production using the dual and triple transfection systems. As shown in Figures 1A and 1C, the VG productivity and percentage of intact vector genome obtained from production using the dual vector transfection system were found to be higher than those obtained from the triple vector transfection system. These data demonstrate that the use of the dual vector transfection system increases rAAV titers compared to the control triple vector transfection system. The various conditions shown in Figures 1A-1C are listed in Table 1.

[0184] To determine whether the increased VG productivity and percentage of intact vector genome obtained from the dual transfection system could be reproduced using different transgene vectors, confirmatory experiments were performed using additional transfection conditions. The transfection conditions were set as described in Table 3, and the elements contained in the various vectors are described in Table 2. In Table 2, vector ratios were based on mass. [Table 3]

[0185] Transfection mixtures for each transfection condition were prepared in appropriately sized conical tubes by adding calculated volumes of vector(s), OptiPro medium, and polyethyleneimine (PEI), all at ambient temperature. Transfection mixtures were added to cells at a concentration of 1 μg DNA / 1E6 cells. Shake flasks were incubated for 72 hours before harvesting. At harvest, cells were lysed and crude lysate samples were collected after centrifugation to remove cell debris for subsequent droplet digital PCR (ddPCR) and capsid analysis by ELISA.

[0186] Figures 2A-C show the VG productivity (Figure 2A), capsid productivity (Figure 2B), and percentage of intact vector genome (Figure 2C) obtained from production using the dual transfection system and the triple transfection system. As shown in Figures 2A and 2C, the VG productivity and percentage of intact vector genome obtained from production using the dual vector transfection system were found to be higher than those obtained from the triple vector transfection system. The productivity improvement of the dual vector transfection system was found to be consistent across at least two different transgene vectors, including either an edited genome containing human genome-specific homology arms (conditions 1 and 2) or an edited genome containing mouse genome-specific homology arms (conditions 3 and 4). The various conditions shown in Figures 2A-C are listed in Table 3.

[0187] Taken together, the data presented in this example demonstrate the efficacy of the dual vector transfection system compared to the triple transfection system, in particular the dual vector transfection system increased crude lysate titers and the percentage of intact vector genomes.

[0188] Example 3: Comparison between dual vector transfection system designs To investigate whether the organization of vector elements in a dual vector transfection system affects productivity, two dual vector transfection system designs were tested. Vector genome (VG) productivity and the percentage of intact vector genomes obtained from production based on each design were evaluated. Dual vector transfection system Design-1 ("Design-1") and Design-2 ("Design-2") differ with respect to which vector the Rep / Cap sequences are present relative to the vector genome and helper sequences. Figures 3A-3B show schematic diagrams of Design-1 (Figure 3A) and Design-2 (Figure 3B). As shown, Design-1 includes a first vector containing Rep / Cap sequences and a transgene ("GOI") and a second vector containing helper sequences (Figure 3A); Design-2 includes a first vector containing a transgene ("GOI") and a second vector containing both helper and Rep / Cap sequences (Figure 3B). Transfection conditions were set up as described in Table 4. [Table 4]

[0189] As shown in Table 4, Design-1 includes a first V4 vector and a second V3 vector. Design-2 includes a first V1 vector and a second V7 vector. VG productivity and percentage of intact vector genome obtained from triple transfection were evaluated as controls. Elements contained in various vectors are listed in Table 2. In Table 4, vector ratios were based on plasmid size (i.e., molar ratio) to take into account different sizes of vectors when comparing dual vector transfection system designs.

[0190] Transfection mixtures for each transfection condition were prepared in appropriately sized conical tubes by adding calculated volumes of vector(s), OptiPro medium, and polyethyleneimine (PEI), all at ambient temperature. Transfection mixtures were added to cells at a concentration of 1 μg DNA / 1E6 cells. Shake flasks were incubated for 72 hours before harvesting. At harvest, cells were lysed and crude lysate samples were collected after centrifugation to remove cell debris for subsequent droplet digital PCR (ddPCR) and capsid analysis by ELISA.

[0191] Figures 4A-C show the VG productivity (Figure 4A), capsid productivity (Figure 4B), and percentage of intact vector genome (Figure 4C) obtained from production using dual transfection system and triple transfection system. As shown in Figures 4A and 4C, the VG productivity and percentage of intact vector genome obtained from production using Design-1 were found to be higher than those obtained from triple transfection system. Furthermore, as shown in Figures 4A and 4C, the calculated VG productivity and percentage of intact vector genome obtained from production using Design-1 were found to be higher than those obtained from production using Design-2. Based on these results, Design-1 was selected for further studies. The various conditions shown in Figures 4A-C are listed in Table 4.

[0192] A third dual vector transfection system design ("Design-3") was tested. The productivity of vector genome (VG) and the percentage of intact vector genome resulting from production based on each of the three designs were evaluated side-by-side. As described above, Design-1 includes a first vector containing a Rep / Cap sequence and a transgene ("GOI"), and a second vector containing a helper sequence (Figure 3A); Design-2 includes a first vector containing a transgene ("GOP") and a second vector containing both a helper sequence and a Rep / Cap sequence (Figure 3B); and Design-3 includes a first vector containing a transgene ("GOI") and a helper sequence, and a second vector containing a Rep / Cap sequence (Figure 3C). Transfection conditions were set as described in Table 5. [Table 5]

[0193] As shown in Table 5, Design-1 includes a first V20 vector and a second V3 vector. Design-2 includes a first V19 vector and a second V7 vector. Design-3 includes a first V21 vector and a second V2 vector. VG productivity and percentage of intact vector genome obtained from triple transfection were evaluated as controls. Elements contained in various vectors are listed in Table 2. In Table 5, vector ratios were mass-based ratios converted from 1:1 (1:1:1) molar ratios.

[0194] Transfection mixtures for each transfection condition were prepared in appropriately sized conical tubes by adding calculated volumes of vector(s), OptiPro medium, and polyethyleneimine (PEI), all at ambient temperature. Transfection mixtures were added to cells at a concentration of 1 μg DNA / 1E6 cells. Shake flasks were incubated for 72 hours before harvesting. At harvest, cells were lysed and crude lysate samples were collected after centrifugation to remove cell debris for subsequent droplet digital PCR (ddPCR) and capsid analysis by ELISA.

[0195] 5A-5C show the VG productivity (FIG. 5A), capsid productivity (FIG. 5B), and percentage of intact vector genome (FIG. 5C) obtained from production using dual and triple transfection systems. As shown in FIG. 5A and FIG. 5C, the VG productivity and percentage of intact vector genome obtained from production using Design-1 were found to be higher than those obtained from the triple transfection system. Furthermore, as shown in FIG. 5A and FIG. 5C, the calculated VG productivity and percentage of intact vector genome obtained from production using Design-1 were found to be higher than those obtained from production using Design-2 and Design-3. These data demonstrate that the use of Design-1 dual vector transfection system increases rAAV titers compared to Design-2 dual vector transfection system, Design-3 dual vector transfection system, and the control triple vector transfection system. The various conditions shown in FIG. 5A-5C are listed in Table 5.

[0196] Example 4: Comparison between dual and triple transfection systems To confirm the increased productivity of Design-1 over triple transfection observed in Example 3, transfection conditions were set up to examine whether the increased efficiency was maintained at a larger scale (2L scale) and whether the increased efficiency of Design-1 extended to different capsids across packaging of rAAV genomes with different transgenes. Transfection conditions were set up as described in Table 6. In Table 6, vector ratios were based on mass. [Table 6-1] [Table 6-2]

[0197] Transfection conditions 1, 2, 3, 4, 5 and 6 were set up as described in Table 6 to examine whether the increased efficacy of Design-1 extends to the packaging of rAAV genomes with different transgenes. Conditions 6 and 7, in addition to examining the efficacy of Design-1 across the packaging of rAAV genomes with different transgenes, also evaluate whether its efficacy extends to the packaging of rAAV genomes with different capsids. Conditions 1 to 5 utilized AAVHSCS15 capsids, condition 6 utilized AAVHSCS17 capsids, and condition 7 utilized AAV2 capsids. VG productivity from triple transfection and the percentage of intact vector genomes were evaluated as controls. The elements contained in the various vectors are described in Table 2.

[0198] Transfection mixtures for each transfection condition were prepared by adding calculated volumes of vector(s), OptiPro medium, and polyethylenimine (PEI) in an appropriately sized transfer assembly, all at ambient temperature. Transfection mixtures were added to cells at a concentration of 1 μg DNA / 1E6 cells. Cells were incubated for 72 hours before harvesting.

[0199] Upon harvest, cells were lysed and crude lysate samples were collected after centrifugation to remove cellular debris for subsequent capsid analysis by droplet digital PCR (ddPCR) and ELISA.

[0200] Figures 6A-6C show the VG productivity (Figure 6A), capsid productivity (Figure 6B), and percentage of intact vector genome (Figure 6C) obtained from production using Design-1 and the control triple transfection system. As shown in Figures 6A and 6C, the VG productivity and percentage of intact vector genome obtained from production using Design-1 were found to be higher than those obtained from the triple transfection system in all conditions tested. Based on these results, an increased efficiency of production using Design-1 over triple transfection was observed across packaging of rAAV with different transgenes into different capsids. The increased productivity of the dual vector transfection system was found to be consistent across five different rAAV genomes, two of which contained edited genomes (conditions 1 and 2). These data demonstrate that the increased rAAV titers obtained using the Design-1 dual vector transfection system over the control triple vector transfection system extend across packaging of rAAV with different transgenes into different capsids. The various conditions shown in Figures 6A-C are listed in Table 6.

[0201] Figures 7A-7C show the VG productivity (Figure 7A), capsid productivity (Figure 7B), and percentage of intact vector genome (Figure 7C) obtained from production utilizing AAV2 capsids using Design-1 and the control triple transfection system (condition 7). As shown in Figures 7A and 7C, the VG productivity and percentage of intact vector genome obtained from production utilizing AAV2 capsids using Design-1 were found to be higher than those obtained from the triple transfection system. The data in Figures 7A-7C were generated from a small-scale production study.

[0202] In a separate experiment, Design-1 was also found to be capable of producing rAAV containing the AAVHSC13 capsid (see U.S. Patent No. 9,803,218, which is incorporated herein in its entirety).

[0203] These data suggest that the improvement in AAV production exhibited by the Design-1 dual plasmid system (compared to the triple plasmid system control) is likely to be generally applicable.

[0204] Example 5: Comparison between dual and triple transfection systems In Examples 3 and 4, an increase in VG productivity and percentage of intact vector genomes measured in crude lysates obtained from production using Design-1 was demonstrated compared to production using the control triple transfection system.

[0205] To confirm that the increased VG productivity and the increased percentage of intact vector genomes were maintained after purification, crude lysates obtained from transfections set up according to those listed in Table 7 were characterized and subsequently purified by affinity and anion exchange chromatography. In Table 7, conditions 1, 2, and 4 were performed according to conditions 2, 3, and 5 in Table 6, respectively (i.e., at 2 L scale), except for condition 3, which was performed at 50 L scale. Lysates produced with different vector ratios were purified separately. Conditions 1-3 each utilize AAVHSCS15 capsids, whereas condition 4 utilized AAVHSCS17 capsids. The amount of intact vector genome obtained from the Design-1 dual plasmid system was expressed as a percentage increase relative to the amount of intact vector genome obtained from the control triple plasmid system as indicated (Table 7 and Figure 8). In Table 7, the vector ratios were based on mass. The elements contained in the various vectors are listed in Table 2. [Table 7]

[0206] The data presented in Figure 8 is based on analytical ultracentrifugation sedimentation velocity (AUC) analysis, a method used to quantify macromolecules based on sedimentation coefficient. AUC was used to determine the percentage of intact vector genomes and capsids lacking vector genomes produced by each Design-1 dual plasmid system relative to the corresponding triple plasmid system control. In Figure 8, for conditions 1 and 2, AUC was performed on purified vectors obtained from each of the Design-1 vector ratios (i.e., 1:2, 1:3, and 1:4 ratios shown in Table 7) to determine the number of intact vector genomes, which were then averaged and presented as a percent increase relative to the corresponding triple plasmid system control. As shown in Figure 8, an increase in the number of intact vector genomes was obtained for each of the four Design-1 dual plasmid systems tested (relative to the number of intact vector genomes obtained from the corresponding triple plasmid system control). These data suggest that the improvement in AAV production exhibited by the Design-1 dual plasmid system (relative to the triple plasmid system control) is likely to be generally applicable and scalable.

[0207] Example 6: Background expression of capsid in a dual transfection system To clarify why Design-1 is superior to other dual plasmid transfection system designs, the level of background capsid expression was determined in Design-1 and compared with the level of background capsid expression in Design-2. The transfection conditions were set as described in Table 8. In Table 8, the vector ratio was based on mass. [Table 8]

[0208] Design-1 and Design-2 were tested with only the Rep / Cap containing vector for each dual design, as shown in Table 8. The same amount of the Rep / Cap containing vector was used alone (e.g., conditions 2 and 4) or as the vector in the dual design (e.g., conditions 1 and 3).

[0209] The level of background capsid production from design-2 (transfection of vector V7 only; condition 2) was found to be the same as the level of background capsid produced from dual transfection of design-2 (transfection of both vectors V1 and V7; condition 1) (Figure 9). As shown in Figure 9, background capsid production from design-1 was less than 1% of the level of background capsid produced by dual transfection of design-1 (compare condition 4 with condition 3).

[0210] Example 7: Large-scale production and quality assessment of AAV from dual transfection and triple transfection systems To examine whether the improved productivity of Design-1 is maintained in larger-scale production, condition 4 in Table 6 was repeated at a 50 L bioreactor scale with a 1:2 vector ratio for Design-1. Consistent with the trends from the shake flask and 2 L bioreactor scales, the 50 L bioreactor results demonstrated a nearly 2-fold increase in VG productivity, comparable capsid production, and twice the calculated intact vector genomes in the crude lysate from Design-1 ("2TFX") compared to the crude lysate from the triple transfection system ("3TFX"; see Table 6 for triple transfection control conditions) (Figures 10A-C). These data demonstrate that the increased rAAV titers obtained using the Design-1 dual vector transfection system compared to the control triple vector transfection system are maintained in larger-scale production.

[0211] Various analytical methods were used to characterize the product quality of AAV vectors obtained from Design-1 and the triple transfection system (Figures 10D-10J). As shown, the purity % (Figure 10D), aggregation rate (Figure 10E), and levels of residual host cell proteins (Figure 10F; BLoQ means below the limit of quantification) all remained constant regardless of transfection method. No deviation was found in the amount of residual host cell DNA (Figure 10G), Rep / Cap (Figure 10H), Ela (Figure 10I), and helper sequences (Figure 10J) packaged in purified AAV vectors obtained from Design-1 compared to those obtained from the triple transfection system.

[0212] Example 8: Biological activity of AAV vectors obtained from dual transfection and triple transfection systems To ensure product comparability between the AAV vectors obtained from Design-1 and the triple transfection system, AAV vectors obtained from the 1:4 vector ratio in Design-1 and the relevant triple transfection control from condition 5 in Table 6 were purified and evaluated for in vivo biological activity. The rAAV genome contains an edited genome expressing phenylalanine hydroxylase (PAH) under the control of a liver-specific promoter flanked by mouse-specific homology arms. The AAV vectors obtained from Design-1 and the triple transfection system were then transfected into mouse models of Pah, a model that exhibits some characteristics of classical phenylketonuria. enu2Mice were injected with 100 mg of VG / kg IgG. The two doses were evaluated as well as a vehicle-only control group. Weekly serum samples were collected and analyzed for phenylalanine (Phe) concentrations. As shown in Figures 11A and 11B, the bioactivity of AAV vectors derived from Design-1 and the triple transfection system, as indicated by the reduction in serum Phe concentrations after administration, was indistinguishable over a 6-week period at both doses of 1E12VG / kg (Figure 11A) and 1E14VG / kg (Figure 11B). Furthermore, at the 6-week time point, quantification of vector genomes in liver and PAH mRNA expression showed a dose-dependent increase in VG transduction and transgene expression, but no significant differences were found between Design-1 and the triple transfection group at each dose (Figures 11C and 11D). Quantification of on-target integration was completed at the 1E14VG / kg dose, demonstrating comparable integration efficiency for AAV vectors produced from either Design-1 or the triple transfection system (Figure 11E).

[0213] Example 9: Vector ratio optimization Various Design-1 vector ratios were tested to see if there was an optimal vector ratio that improved productivity. Transfection was set up as described in Example 1 for small-scale production.

[0214] Figures 12A-C show VG productivity (Figure 12A), capsid productivity (Figure 12B), and percentage of intact vector genome (Figure 12C) resulting from production under condition 1, where the indicated V3:V12 vector ratios were tested, at various levels of total DNA transfected (x-axis). Elements contained in V3 and V12 are shown in Table 2. As shown in Figures 12A-C, improved VG and capsid productivity was achieved with V3:V12 vector ratios of 1:0.3 to 1:1, using 0.6 to 1 μg of total DNA transfected per 1E6 cells.

[0215] Figures 13A-13C show VG productivity (Figure 13A), capsid productivity (Figure 13B), and percentage of intact vector genome (Figure 13C) resulting from production in condition 2, testing the indicated V3:V8 ratios, at various levels of total DNA transfected (x-axis). Elements contained in V3 and V8 are shown in Table 2. As shown in Figures 13A-13C, improved VG and capsid productivity was achieved with V3:V8 vector ratios of 1:0.6 to 1:1, using 0.6 to 1 μg of total DNA transfected per 1E6 cells. These data demonstrate that increased rAAV titers are achieved using these vector ratios and levels of total DNA transfected.

[0216] Example 10: Evaluation of multiple capsid serotypes using dual plasmid transfection To investigate whether the improved productivity of Design-1 is maintained across other AAV capsid serotypes, AAV vectors produced from either Design-1 or the triple transfection system were tested utilizing AAV capsid serotypes AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10, and AAVrh74. Transfections were set up as described in Example 1 for 2L bioreactor production. Transfection conditions were set up as described in Table 9. [Table 9]

[0217] 14A-14C show the VG productivity (FIG. 14A), capsid productivity (FIG. 14B) and percentage of intact vector genome (FIG. 14C) obtained from production under the conditions shown in Table 9. As shown in FIG. 14A, the improvement in VG productivity obtained from production using Design-1 is maintained across all AAV capsid serotypes tested compared to the corresponding triple transfection system control. As shown in FIG. 14B, the capsid productivity obtained from production using Design-1 is improved or maintained compared to the corresponding triple transfection system control. As shown in FIG. 14C, the percentage of intact vector genome obtained from production using Design-1 is improved or maintained compared to the corresponding triple transfection system control. These data demonstrate that the increased rAAV titers obtained using the Design-1 dual vector transfection system are across the various AAV capsid serotypes compared to the control triple vector transfection system.

[0218] Example 11: Scalability of dual plasmid to 2000 L Example 7 showed that the improved productivity of Design-1 was maintained at the 50 L bioreactor scale. Results from the 50 L bioreactor demonstrated an almost 2-fold increase in VG productivity in the crude lysate obtained from Design-1 compared to the crude lysate obtained from the triple transfection system control.

[0219] To examine whether the improved VG productivity of Design-1 is scalable, the productivity at 50L bioreactor scale was compared with that at 2000L bioreactor scale. Transfection was set up as described in Example 1 for 2L bioreactor production, except that cells were inoculated into 50L and 2000L bioreactors. Cells were transfected at a density of 3.6E6 to 5E6 cells / mL. Transfection conditions for 50L and 2000L bioreactors were set up according to the conditions listed in Table 10. [Table 10]

[0220] Figure 15 shows that 50L and 2000L bioreactor scales achieve comparable VG productivity. These data demonstrate the scalability of the Design-1 dual plasmid transfection system.

[0221] Further embodiments of the present invention are described in the following clauses.

[0222] 1. A first nucleic acid vector comprising: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, the first nucleic acid vector being free of helper virus genes.

[0223] 2. The nucleic acid vector of clause 1, comprising in the 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein, the nucleic acid vector being free of helper virus genes.

[0224] 3. The nucleic acid vector of clause 1, comprising in the 5' to 3' direction: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein.

[0225] 4. The nucleic acid vector according to any one of clauses 1 to 3, wherein said nucleic acid vector is a DNA plasmid or a DNA minimal vector.

[0226] 5. A recombinant AAV (rAAV) packaging system comprising: (i) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein; and (ii) a second nucleic acid vector comprising helper virus genes.

[0227] 6. The packaging system of clause 5, wherein the first nucleic acid vector comprises, in a 5' to 3' direction, a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome comprising a transgene; and a third nucleotide sequence encoding an AAV capsid protein.

[0228] 7. The packaging system of clause 5 or 6, wherein the first nucleic acid vector is a DNA plasmid or a DNA minimal vector.

[0229] 8. The packaging system of any one of clauses 5 to 7, wherein the second nucleic acid vector is a DNA plasmid or a DNA minimal vector.

[0230] 9. The nucleic acid vector or packaging system according to any one of clauses 1 to 8, wherein said transgene encodes a polypeptide.

[0231] 10. The nucleic acid vector or packaging system of any one of clauses 1 to 8, wherein the transgene encodes a miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, lncRNA, ribozyme, or mRNA.

[0232] 11. The nucleic acid vector or packaging system of any one of clauses 1 to 8, wherein the transgene encodes a protein selected from the group consisting of phenylalanine hydroxylase (PAH), glucose-6-phosphatase (G6Pase), iduronate-2-sulfatase (I2S), arylsulfatase A (ARSA), and frataxin (FXN).

[0233] 12. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the rAAV genome further comprises a transcriptional regulatory element operably linked to the transgene.

[0234] 13. The nucleic acid vector or packaging system of clause 12, wherein the transcriptional regulatory elements comprise promoter elements and / or intron elements.

[0235] 14. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the rAAV genome further comprises a polyadenylation sequence.

[0236] 15. The nucleic acid vector or packaging system of clause 14, wherein the polyadenylation sequence is 3' to the transgene.

[0237] 16. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:71, 85, 86, 87, or 88.

[0238] 17. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the rAAV genome further comprises a 5' inverted terminal repeat (5'ITR) nucleotide sequence 5' of the transgene and a 3' inverted terminal repeat (3'ITR) nucleotide sequence 3' of the transgene.

[0239] 18. The nucleic acid vector or packaging system according to clause 17, wherein the 5' ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39, 41 or 42, and / or the 3' ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 40, 43 or 44.

[0240] 19. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 75, 78, 80, 82 or 84.

[0241] 20. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the AAV Rep protein is a wild-type Rep protein or a variant thereof.

[0242] 21. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the AAV Rep protein is an AAV2 Rep protein or a variant thereof.

[0243] 22. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the first nucleotide sequence further comprises a transcriptional regulatory element operably linked to the AAV Rep protein coding sequence.

[0244] 23. The nucleic acid vector or packaging system of clause 22, wherein said transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter or a native promoter.

[0245] 24. The nucleic acid vector or packaging system of clause 23, wherein the promoter is selected from the group consisting of a P5 promoter, a P19 promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter, and a rapamycin-inducible promoter.

[0246] 25. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVRh32.33, AAVrh74, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, and PHP.S.

[0247] 26. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0248] 27. The amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I; and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R. 27. The nucleic acid vector or packaging system of clause 26, wherein: the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

[0249] 28. (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is H, (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.

[0250] 29. The nucleic acid vector or packaging system of clause 27, wherein the AAV capsid protein comprises the amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0251] 30. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0252] 31. The amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO:16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO:16 is S; and the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I. 31. The nucleic acid vector or packaging system of clause 30, wherein: the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

[0253] 32. (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO:16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO:16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is H, is R and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO:16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C.

[0254] 33. The nucleic acid vector or packaging system of clause 31, wherein the AAV capsid protein comprises the amino acid sequence of amino acids 138 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0255] 34. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0256] 35. The amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO:16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO:16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO:16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO:16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO:16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO:16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO:16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO:16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO:16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 35. The nucleic acid vector or packaging system of clause 34, wherein the amino acid in the capsid protein is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

[0257] 36. (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y; (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (f) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is A; 36. The nucleic acid vector or packaging system of clause 35, wherein: (a) the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is H, (b) the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO:16 is N, (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and (d) the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO:16 is M; (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R and (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:16 is A and (i) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO:16 is I, (i) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:16 is R, and (i) the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO:16 is C.

[0258] 37. The nucleic acid vector or packaging system of clause 35, wherein the AAV capsid protein comprises the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0259] 38. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the third nucleotide sequence further comprises a transcriptional regulatory element operably linked to the AAV capsid protein coding sequence.

[0260] 39. The nucleic acid vector or packaging system of clause 38, wherein the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter or a native promoter.

[0261] 40. The nucleic acid vector or packaging system of clause 39, wherein the promoter is selected from the group consisting of a P40 promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter, and a rapamycin-inducible promoter.

[0262] 41. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the first nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 73 or 77.

[0263] 42. The nucleic acid vector or packaging system of any of the preceding clauses, wherein the second nucleotide sequence comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 71, 75, 78, 80, 82, 84, 85, 86, 87, or 88.

[0264] 43. The first nucleotide sequence comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59; and the second nucleotide sequence comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% identical to the nucleotide sequence set forth in SEQ ID NO: 71, 75, 78, 80, 82, 84, 85, 86, 87, or 88. , 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of amino acids 203 to 736, 138 to 736 and / or 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 or 17.

[0265] 44. The nucleic acid vector or packaging system according to clause 43, wherein the first nucleic acid vector comprises, in the 5' to 3' direction: the first nucleotide sequence, the second nucleotide sequence; and the third nucleotide sequence.

[0266] 45. The packaging system of any one of clauses 5 to 44, wherein said helper virus gene is derived from a helper virus selected from the group consisting of adenovirus, herpesvirus, poxvirus, cytomegalovirus, and baculovirus.

[0267] 46. ​​The packaging system of any one of clauses 5 to 45, wherein said helper virus gene is an RNA gene derived from an adenovirus selected from the group consisting of El, E2, E4 and VA.

[0268] 47. The packaging system of any one of clauses 5-46, wherein the second nucleic acid vector further comprises a transcriptional regulatory element operably linked to the helper virus gene.

[0269] 48. The packaging system of clause 47, wherein the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter or a native promoter.

[0270] 49. The packaging system of clause 48, wherein the promoter is selected from the group consisting of an RSV LTR promoter, a CMV immediate early promoter, an SV40 promoter, a dihydrofolate reductase promoter, a cytoplasmic β-actin promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter and a rapamycin-inducible promoter.

[0271] 50. The packaging system of any one of clauses 5 to 49, wherein the second nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 60, 61, or 62.

[0272] 51. The packaging system of any one of clauses 5 to 50, wherein the second nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:63.

[0273] 52. The packaging system of any one of clauses 5 to 45, wherein said helper virus gene is a gene from a herpesvirus selected from the group consisting of UL5 / 8 / 52, ICP0, ICP4, ICP22 and UL30 / UL42.

[0274] 53. The packaging system of clause 52, wherein said second nucleic acid vector further comprises a transcriptional regulatory element operably linked to said helper virus gene.

[0275] 54. The packaging system of clause 53, wherein the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter or a native promoter.

[0276] 55. The packaging system of clause 54, wherein the promoter is selected from the group consisting of an RSV LTR promoter, a CMV immediate early promoter, an SV40 promoter, a dihydrofolate reductase promoter, a cytoplasmic β-actin promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, a RU486-inducible promoter and a rapamycin-inducible promoter.

[0277] 56. A host cell comprising a nucleic acid vector according to any one of clauses 1 to 4 or clauses 9 to 44, or a packaging system according to any one of clauses 5 to 55.

[0278] 57. The host cell according to clause 56, wherein the host cell is a mammalian cell.

[0279] 58. The host cell according to clause 57, wherein the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HEK293F cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, HeLa cells, NIH3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

[0280] 59. The host cell according to clause 57 or 58, wherein the mammalian cell is a HEK293 cell.

[0281] 60. A method for the recombinant preparation of rAAV, comprising introducing into a mammalian cell a packaging system described in any one of clauses 5 to 55 under conditions for producing rAAV.

[0282] 61. The method of claim 60, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is selected from the group consisting of 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:3, or 1:4.

[0283] 62. The method of clause 60 or 61, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:2.

[0284] 63. The method according to clause 60 or 61, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.2 to 1:1.

[0285] 64. The method of clause 63, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.6.

[0286] 65. The method of clause 63, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:0.8.

[0287] 66. The method of clause 63, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is 1:1.

[0288] 67. The method of any one of clauses 60 to 66, wherein the method comprises introducing 0.1 to 4 μg of DNA of the packaging system per 1E6 cell.

[0289] 68. The method of any one of clauses 60 to 67, wherein the method comprises introducing 0.5 to 1 μg of DNA of the packaging system per 1E6 cell.

[0290] 69. The method of any one of clauses 60 to 68, wherein the method comprises introducing 0.6, 0.7, 0.8, 0.9 or 1 μg of DNA / 1E6 cell of the packaging system.

[0291] 70. The method of any one of clauses 60 to 68, wherein the method comprises introducing 0.75 μg DNA / 1E6 cell of the packaging system.

[0292] 71. The method of any one of clauses 60 to 70, which results in increased rAAV titer compared to a method comprising producing rAAV using a mammalian cell comprising: (i) a first vector comprising a nucleotide sequence encoding the AAV Rep protein and the AAV capsid protein; (ii) a second vector comprising the rAAV genome; and (iii) a third vector comprising one or more of the helper virus genes.

[0293] 72. The method of any one of clauses 60 to 70, which results in an increased percentage of intact vector genomes compared to a method comprising producing rAAV using a mammalian cell comprising: (i) a first vector comprising nucleotide sequences encoding the AAV Rep proteins and the AAV capsid proteins; (ii) a second vector comprising the rAAV genome; and (iii) a third vector comprising one or more of the helper virus genes.

[0294] 73. The method of any one of clauses 60-72, wherein said mammalian cells are selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HEK293F cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, HeLa cells, NIH3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

[0295] 74. The method of any one of clauses 60 to 73, wherein the mammalian cell is a HEK293 cell.

[0296] 75. The method of any one of clauses 60 to 74, wherein the mammalian cells are incubated in cell culture.

[0297] 76. A population of host cells defined in any one of clauses 56 to 59, wherein said host cells are provided in a cell culture.

[0298] 77. The method of clause 75 or the population of host cells of clause 76, wherein the cell culture has a volume of at least 2 litres, at least 50 litres or at least 2000 litres. *** The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the appended claims. All references (e.g., publications, or patents, or patent applications) cited herein are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the scope of the following claims.

Claims

1. a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome containing a transgene; a third nucleotide sequence encoding an AAV capsid protein, and a first nucleic acid vector that does not contain a helper virus gene.

2. The nucleic acid vector according to claim 1, wherein the nucleic acid vector is a DNA plasmid or a DNA minimal vector.

3. (i) a first nucleic acid vector comprising: a first nucleotide sequence encoding an AAV Rep protein; a second nucleotide sequence comprising a recombinant AAV (rAAV) genome containing a transgene; and a third nucleotide sequence encoding an AAV capsid protein, and (ii) a second nucleic acid vector containing a helper virus gene, a recombinant AAV (rAAV) packaging system.

4. The packaging system according to claim 3, wherein the first nucleic acid vector is a DNA plasmid or a DNA minimal vector.

5. The packaging system according to claim 3, wherein the second nucleic acid vector is a DNA plasmid or a DNA minimal vector.

6. The nucleic acid vector or packaging system according to any one of the preceding claims, wherein the rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence at the 5' of the transgene and a 3' inverted terminal repeat (3' ITR) nucleotide sequence at the 3' of the transgene.

7. The nucleic acid vector or packaging system according to claim 6, wherein the length of both the 5' and 3' ITR nucleotide sequences is 145 base pairs. **Claim 8**: The nucleic acid vector or packaging system according to claim 6, wherein the 5' ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39, 41, or 42, and / or the 3' ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 40, 43, or 44. **Claim 9** The nucleic acid vector according to claim 1 or 2, or the packaging system according to any one of claims 3 to 5, wherein the AAV Rep protein is a wild-type Rep protein or a variant thereof. **Claim 10** The nucleic acid vector according to claim 1 or 2, or the packaging system according to any one of claims 3 to 5, wherein the AAV Rep protein is an AAV2 Rep protein or a variant thereof. **Claim 11** The packaging system according to any one of claims 3 to 5, wherein the helper virus gene is derived from a helper virus selected from the group consisting of adenovirus, herpesvirus, poxvirus, cytomegalovirus, and baculovirus. **Claim 12** The packaging system according to any one of claims 3 to 5, wherein the helper virus gene is an RNA gene derived from an adenovirus selected from the group consisting of El, E2, E4, and VA. **Claim 13** The packaging system according to any one of claims 3 to 5, wherein the second nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 60, 61, or 62. **Claim 14** The packaging system according to any one of claims 3 to 5, wherein the second nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:

63.

15. A host cell comprising the nucleic acid vector according to claim 1 or 2, or the packaging system according to any one of claims 3 to 5.

16. The host cell according to claim 15, wherein the host cell is a mammalian cell.

17. The host cell according to claim 16, wherein the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HEK293F cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78BsT cells, HeLa cells, NIH3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, B-W cells, L-M cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

18. The host cell according to claim 16 or 17, wherein the mammalian cell is HEK293 cells.

19. A method for the recombinant production of rAAV, the method comprising introducing the packaging system according to any one of claims 3 to 5 into a mammalian cell under conditions for producing the rAAV.

20. The method according to claim 19, wherein the ratio of the first nucleic acid vector to the second nucleic acid vector, or the ratio of the second nucleic acid vector to the first nucleic acid vector, is selected from the group consisting of 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:3, or 1:

4.

21. The method according to claim 19, wherein the method comprises introducing 0.1 to 4 μg of DNA / 1E6 cells of the packaging system.

22. The method according to claim 19, wherein the mammalian cell is provided in a cell culture.

23. A population of the host cells according to claim 15 provided in a cell culture.

24. The method according to claim 22 or the population of host cells according to claim 23, wherein the cell culture has a volume of at least 2 liters, at least 50 liters, or at least 2000 liters.

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