Compositions and methods for recombinant AAV production

Abstract

This specification provides recombinant polynucleotides and plasmids suitable for use in the production of recombinant AAV particles. This specification also provides methods for producing rAAV particles.

Description

Technical Field

[0001] The present disclosure relates to recombinant polynucleotides and their use in methods for generating recombinant adeno-associated virus (rAAV) particles.

[0002] Cross - Reference to Related Applications This application claims the benefit of U.S. Patent Application No. 63 / 494,859, filed Apr. 7, 2023, which is hereby incorporated by reference in its entirety.

[0003] Reference to Electronically Submitted Sequence Listing The content of the sequence listing electronically submitted in this application (name: 67282001_Sequence_Listing.xml, size: 2,211,668 bytes, date of creation: Apr. 4, 2024) is hereby incorporated by reference in its entirety into this specification.

Background Art

[0004] Background Recombinant adeno - associated virus (AAV) - based vectors are currently the most widely used developing gene therapy products. The reasons for the preference for the use of rAAV vector systems are, in part, the absence of diseases associated with wild - type viruses, the ability of AAV transduction in both non - dividing and dividing cells, and the fact that long - term robust transgene expression has been observed in clinical trials, which indicates great potential for delivery in gene therapy applications. In addition, various natural and recombinant rAAV vector serotypes specifically target various tissues, organs, and cells and help to avoid any existing immunity to the vector, thus expanding the therapeutic applications of AAV - based gene therapy. Before recombinant virus particles can be more widely adopted for late - stage clinical and commercial use, there is a need to develop new methods for large - scale production of recombinant virus particles.

[0005] The triple plasmid translocation system in HEK293 cells is well-established and commonly used for clinical and commercial production. In this system, one plasmid (often referred to as the trans plasmid) contains the Rep and Cap genes and encodes proteins for viral replication and capsid formation. A second plasmid (often referred to as the helper plasmid) encodes essential adenovirus helper genes (E4, E2A, and virus-associated (VA)RNA), and a third plasmid (often referred to as the cis plasmid) contains an expression cassette with two reverse-terminal repeats (ITRs) adjacent to each other, which is incorporated into rAAV as its genome. Additional helper genes E1A and E1B are endogenously expressed by HEK293 cells. The E1A protein enhances Rep protein expression by transactivating the P5 and P19 promoters, and the Rep protein initiates AAV replication.

[0006] Reducing the number of plasmids used for transient translocation from three to two or even one improves translocation efficiency, simplifies the translocation process, lowers the cost of goods (COG) for manufacturing GMP plasmids, and further shortens the timeline for generating new rAAVs. Triple plasmid systems offer several advantages compared to using helper viruses to generate rAAV particles. These advantages include shorter GMP plasmid manufacturing times, faster deployment to clinical settings, and the absence of adenovirus in the process. However, the translocation efficiency of each plasmid remains a limiting factor. This is because triple plasmid systems require cells to simultaneously take up three plasmids to generate the virus. Several research groups have attempted to create two-plasmid systems to simplify the translocation process, lower the cost of goods (COG) for manufacturing GMP plasmids, and further shorten the timeline for generating new rAAVs. The first two-plasmid expression system was developed by Grimm et al., who designed a packaging / helper plasmid pDG to include all genes from both the trans plasmid and the helper plasmid in a single plasmid. This novel plasmid contained AAV-2 rep and cap, as well as adenovirus E4, E2A, and VA RNA genes (required for AAV replication, capsid formation, and helper function). Furthermore, the p5 promoter driving Rep expression in the trans plasmid was replaced with a mouse mammary tumor virus (MMTV) promoter to weaken Rep protein expression. This new system showed a 10-fold higher titer than conventional methods using helper viruses. Grimm et al., Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum Gene Ther. 1998;9(18):2745-2760. (Non-patent document 1).Tang et al. further modified the backbone of the pDG plasmid to create a pQT packaging system, which included the rep, cap, and essential adenovirus helper genes necessary for AAV generation. They compared the pQT system with a conventional triple transfection system using different AAV serotypes, including AAV1, AAV5, AAV8, and AAV9, and found that the pQT system yielded higher production of AAV1 and AAV5 than the triple transfection system. Tang et al., “Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus.” Biores Open Access.9(2020):219-28.(Non-Patent Literature 2).

[0007] Because the pDG and pQT systems were designed by combining all the genes of the trans and helper plasmids, the plasmid size exceeds 20 Kb, making plasmid production extremely difficult. In addition, the large plasmid size reduces the translocation efficiency during transient translocation. Therefore, in this art, there is a need to improve the productivity and yield of methods for large-scale production of rAAV particles by providing improved helper and packaging plasmids. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Grimm et al., Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum Gene Ther.1998;9(18):2745-2760. [Non-Patent Document 2] Tang et al., “Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus.” Biores Open Access.9(2020):219-28. [Overview of the project]

[0009] overview In one embodiment, the present disclosure relates to an isolated recombinant polynucleotide, a) A first nucleotide sequence encoding adenovirus E2A DNA-binding protein (DBP), which is functionally bound to the first promoter, b) A second nucleotide sequence encoding the adenovirus E4 polypeptide, which is functionally bound to the second promoter, c) The third nucleotide sequence encoding adenovirus VA RNA I, d) The fourth nucleotide sequence encoding the parvovirus p5 promoter and the fifth nucleotide sequence encoding the AAV rep gene and the AAV cap gene. The isolated recombinant polynucleotide provides an isolated recombinant polynucleotide containing a parvovirus p5 promoter which is actionably bound to the AAV rep gene and controls the expression of the rep78 and rep68 gene products. Optionally, the isolated recombinant polynucleotide does not contain nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursors. In some embodiments, the isolated recombinant polynucleotide further comprises a sixth nucleotide sequence encoding a recombinant viral genome, which includes at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product, actionably bound to a sequence that directs the expression of the gene product in a target cell.

[0010] In one embodiment, the present disclosure provides a host cell comprising an isolated recombinant polynucleotide as described herein.

[0011] In one embodiment, the present disclosure provides a method for generating isolated recombinant polynucleotides as described herein, comprising incubating host cells as described herein under preferred conditions.

[0012] In one embodiment, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising culturing cells capable of generating rAAV particles under conditions that enable the generation of rAAV particles, wherein the cells contain recombinant polynucleotides as described herein.

[0013] In one embodiment, the present disclosure provides a method for generating rAAV particles, comprising: a) preparing a cell culture containing cells; b) introducing a recombinant polynucleotide described herein into the cells; and c) maintaining the cell culture under conditions that enable the generation of rAAV particles.

[0014] In some embodiments, this disclosure provides the following: [1.] Isolated recombinant polynucleotides, a) A first nucleotide sequence encoding adenovirus E2A DNA-binding protein (DBP), which is functionally bound to the first promoter, b) A second nucleotide sequence encoding the adenovirus E4 polypeptide, which is functionally bound to the second promoter, c) The third nucleotide sequence encoding adenovirus VA RNA I, d) A fourth nucleotide sequence encoding the parvovirus p5 promoter, and a fifth nucleotide sequence encoding the AAV rep gene and the AAV cap gene, wherein the parvovirus p5 promoter is capablely bound to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. Includes, Optionally, the isolated recombinant polynucleotide is one which does not contain nucleotide sequences encoding an adenovirus ITR sequence, an L3 23K endoprotease, an L5 pVI / fiber, and / or an L4 pVIII / hexone-related precursor. [2.] The isolated recombinant polynucleotide according to [1], wherein the nucleotide sequence encoding the adenovirus E2A DBP and the nucleotide sequence encoding the adenovirus E4 polypeptide are in opposite 5'→3' directions. [3.] An isolated recombinant polynucleotide according to [1] or [2], wherein the nucleotide sequence encoding the adenovirus E2A DBP has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1 or 60. [4.] An isolated recombinant polynucleotide according to any one of [1] to [3], wherein the adenovirus E2A DBP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 45. [5.] The E4 polypeptide comprises E4 ORF6 and ORF7, an isolated recombinant polynucleotide according to any one of [1] to [4]. [6.] The isolated recombinant polynucleotide according to [5], wherein the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 8 or 61. [7.] The isolated recombinant polynucleotide according to [5] or [6], wherein the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 46, and the adenovirus E4 ORF7 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 120. [8.] The E4 polypeptide comprises E4 ORF6, an isolated recombinant polynucleotide according to any one of [1] to [4]. [9.] The isolated recombinant polynucleotide according to [8], wherein the nucleotide sequence encoding adenovirus E4 ORF6 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 6. [10.] The isolated recombinant polynucleotide according to [9] or

[10] , wherein the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 46. [11.] An isolated recombinant polynucleotide according to any one of [1] to

[10] , wherein the nucleotide sequence encoding adenovirus VA RNA I has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 54. [12.] An isolated recombinant polynucleotide according to any one of [1] to

[10] , wherein the nucleotide sequence encoding adenovirus VA RNA I encodes VA RNA I and VA RNA II, and optionally comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9. [13.] An isolated recombinant polynucleotide according to any one of [1] to

[12] , wherein the first promoter and the second promoter are different promoters. [14.] An isolated recombinant polynucleotide as described in any one of [1] to

[13] , wherein the first promoter is the adenovirus E2A promoter. [15.] The isolated recombinant polynucleotide according to

[14] , wherein the adenovirus E2A promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with respect to SEQ ID NO: 2. [16.] An isolated recombinant polynucleotide according to any one of [1] to

[15] , comprising a nucleotide sequence wherein the first nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 3, 4, 23, 24, 273-275, or 276. [17.] An isolated recombinant polynucleotide as described in any one of [1] to

[16] , wherein the second promoter is the adenovirus E4 promoter. [18.] The isolated recombinant polynucleotide according to

[17] , wherein the adenovirus E4 promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 5. [19.] An isolated recombinant polynucleotide according to any one of [1] to

[18] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10 or 11. [20.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , wherein the isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexon assembly polypeptide comprising a N-terminal deletion, and the E2A DBP, and the N-terminal deletion of the L4 100k / hexon assembly polypeptide corresponds to the nucleotide sequence of SEQ ID NO: 21. [21.] The isolated recombinant polynucleotide according to

[20] , wherein the nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexon assembly polypeptide comprising a N-terminal deletion, and the E2A DBP comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22. [22.] An isolated recombinant polynucleotide according to any one of [1] to

[18] , wherein the isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexon assembly polypeptide having a mutation in its start codon, and the E2A DBP. [23.] The isolated recombinant polynucleotide according to

[22] , wherein the nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide, the promoter, the L4 100k / hexone assembly polypeptide having a mutation in its start codon, and the E2A DBP comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 23. [24.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , wherein the isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexone assembly polypeptide having an N-terminal deletion, and the E2A DBP, wherein the N-terminal deletion of the L4 100k / hexone assembly polypeptide includes the start codon of the L4 100k / hexone assembly but does not include the start codon of the L4 22K / 33K polypeptide. [25.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , wherein the isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexone assembly polypeptide having an N-terminal deletion, and the E2A DBP, and all or part of the L4 100k / hexone assembly polypeptide is deleted without disruption of the L4 22K / 33K start codon. [26.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , comprising a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexon assembly polypeptide comprising an N-terminal deletion, and the E2A DBP, wherein the N-terminal deletion of the L4 100k / hexon assembly starts at the start codon of the L4 100k / hexon assembly and ends immediately adjacent to the L4 22K / 33K promoter. [27.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 25-34, 56, 57, 106-109, 122-130, or 131. [28.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260. [29.] The isolated recombinant polynucleotide according to any one of [1] to

[18] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 265 or 266. [30.] The isolated recombinant polynucleotide according to any one of [1] to

[29] , wherein the parvovirus p5 promoter is the AAV p5 promoter. [31.] The isolated recombinant polynucleotide according to

[30] , wherein the AAV p5 promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 62. [32.] An isolated recombinant polynucleotide according to any one of [1] to

[31] , wherein the AAV rep gene and the AAV cap gene have the same serotype. [33.] An isolated recombinant polynucleotide according to any one of [1] to

[31] , wherein the AAV rep gene and the AAV cap gene have different serotypes. [34.] An isolated recombinant polynucleotide according to any one of [1] to

[31] , wherein the AAV rep gene comprises the AAV2 rep gene. [35.] The isolated recombinant polynucleotide according to

[34] , wherein the AAV rep gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 63. [36.] Said AAV The cap gene is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh1 0, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tY Isolated recombinant polynucleotides as described in any one of [1] to

[35] , comprising a serotype selected from the group consisting of F, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. [37.] The isolated recombinant polynucleotide described in

[36] , wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. [38.] The isolated recombinant polynucleotide according to

[36] , wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV8 or AAV9 serotypes. [39.] The isolated recombinant polynucleotide described in

[36] , comprising the AAV cap gene AAV2 serotype. [40.] The isolated recombinant polynucleotide according to

[39] , wherein the AAV2 cap gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 64. [41.] The isolated recombinant polynucleotide described in

[36] , comprising the AAV cap gene AAV6 serotype. [42.] The isolated recombinant polynucleotide according to

[41] , wherein the AAV6 cap gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 65. [43.] The isolated recombinant polynucleotide described in

[36] , comprising the AAV cap gene AAV8 serotype. [44.] The isolated recombinant polynucleotide according to

[43] , wherein the AAV8 cap gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 66. [45.] The isolated recombinant polynucleotide described in

[36] , comprising the AAV cap gene AAV9 serotype. [46.] The isolated recombinant polynucleotide according to

[45] , wherein the AAV9 cap gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 67. [47.] An isolated recombinant polynucleotide according to any one of [1] to

[46] , wherein the sequence encoding the cap gene contains one or more mutations that disrupt the expression of the mAAP polypeptide. [48.] The isolated recombinant polynucleotide according to

[47] , wherein the one or more mutations that disrupt the expression of the mAAP polypeptide include one or more nonsense mutations in the mAAP ORF. [49.] The isolated recombinant polynucleotide according to

[47] , wherein the one or more mutations that disrupt the expression of the mAAP polypeptide include a mutation in the start codon of the mAAP ORF. [50.] An isolated recombinant polynucleotide according to any one of [1] to

[46] , wherein the sequence encoding the cap gene comprises the nucleotide sequence of SEQ ID NOs. 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, or 261. [51.] An isolated recombinant polynucleotide according to any one of [1] to

[50] , wherein the fifth nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 68-70 or 71. [52.] An isolated recombinant polynucleotide according to any one of [1] to

[50] , wherein the fifth nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs: 167, 172, 187, 192, 197, 202, 207, 212, 217, 222, 227, 232, 237, 242, 247, 252, 257, or 262. [53.] An isolated recombinant polynucleotide as described in any one of [1] to

[52] , wherein the p5 promoter is located approximately 10 to 10,000 nucleotides upstream from the start codon of the AAV rep. [54.] An isolated recombinant polynucleotide according to any one of [1] to

[52] , wherein the p5 promoter is located approximately 5,000 to 10,000 nucleotides upstream from the start codon of the AAV rep. [55.] An isolated recombinant polynucleotide according to any one of [1] to

[52] , wherein the p5 promoter is located approximately 1,000 to 5,000 nucleotides upstream from the start codon of the AAV rep. [56.] The isolated recombinant polynucleotide described in

[53] , wherein the p5 promoter is located approximately 1,000 to 4,000 nucleotides upstream from the start codon of the AAV rep. [57.] The isolated recombinant polynucleotide described in

[53] , wherein the p5 promoter is located approximately 1,000 to 3,000 nucleotides upstream from the start codon of the AAV rep. [58.] The isolated recombinant polynucleotide described in

[53] , wherein the p5 promoter is located approximately 2,000 to 5,000 nucleotides upstream from the start codon of the AAV rep. [59.] The isolated recombinant polynucleotide described in

[53] , wherein the p5 promoter is located approximately 2,000 to 4,000 nucleotides upstream from the start codon of the AAV rep. [60.] The isolated recombinant polynucleotide described in

[53] , wherein the p5 promoter is located approximately 2,000 to 3,000 nucleotides upstream from the start codon of the AAV rep. [61.] An isolated recombinant polynucleotide according to any one of [1] to

[52] , wherein the first, second, and / or third nucleotide sequences are located between the p5 promoter upstream of the start codon of the AAV rep and the start codon of the AAV rep. [62.] An isolated recombinant polynucleotide according to any one of [1] to

[52] , wherein the first, second, and third nucleotide sequences are located between the p5 promoter upstream of the start codon of the AAV rep and the start codon of the AAV rep. [63.] An isolated recombinant polynucleotide according to any one of [1] to

[52] , wherein the first, second, or third nucleotide sequence is located between the p5 promoter upstream of the start codon of the AAV rep and the start codon of the AAV rep. [64.] An isolated recombinant polynucleotide as described in any one of [1] to

[63] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence numbers 72-86, 118, 159-161, 168, 173, 178, 183, 188, 193, 198, 203, 208, 213, 218, 223, 228, 233, 238, 243, 248, 253, 558, or 263. [65.] An isolated recombinant polynucleotide according to any one of [1] to

[63] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 76 or 77. [66.] An isolated recombinant polynucleotide as described in any one of [1] to

[63] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence numbers 87-101, 119, 162-164, 169, 174, 179, 184, 189, 194, 199, 204, 209, 214, 219, 224, 229, 234, 239, 244, 249, 254, 259, or 264. [67.] An isolated recombinant polynucleotide according to any one of [1] to

[63] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 91 or 92. [68.] The isolated recombinant polynucleotide according to any one of [1] to

[67] , wherein the isolated recombinant polynucleotide is a plasmid containing a bacterial origin of replication and a selection marker gene. [69.] The isolated recombinant polynucleotide according to

[68] , wherein the bacterial replication origin is ColE1. [70.] The isolated recombinant polynucleotide according to

[68] or

[56] , wherein the selection marker gene is a drug resistance gene. [71.] The isolated recombinant polynucleotide described in

[70] , wherein the selection marker gene is a kanamycin resistance gene. [72.] An isolated recombinant polynucleotide according to any one of [1] to

[71] , wherein the bacterial origin of replication or the selection marker gene is located between the fourth nucleotide and the fifth nucleotide, such that the transcription initiated by the p5 promoter traverses the bacterial origin of replication or the selection marker gene before reaching the AAV rep gene. [73.] An isolated recombinant polynucleotide according to any one of [1] to

[71] , wherein the bacterial origin of replication and the selection marker gene are located between the fourth nucleotide and the fifth nucleotide, such that the transcription initiated by the p5 promoter traverses the bacterial origin of replication and the selection marker gene, respectively, before reaching the AAV rep gene. [74.] An isolated recombinant polynucleotide according to any one of [1] to

[73] , comprising a sixth nucleotide sequence encoding a recombinant viral genome, the sixth nucleotide sequence comprising at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding the gene product, which is competently bound to a sequence that directs the expression of the gene product in a target cell. [75.] The isolated recombinant polynucleotide according to

[74] , wherein the AAV rep gene, the AAV cap gene, and the non-AAV nucleic acid sequence encoding the gene product are transcribed in the same direction. [76.] An isolated recombinant polynucleotide according to

[74] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 102, 103, 267, or 268, excluding the nucleotide residues corresponding to the nucleotide sequence encoding the recombinant viral genome. [77.] An isolated recombinant polynucleotide according to

[74] , comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 104 or 105, excluding residues 9,537-14,270 of SEQ ID NO: 104 and residues 9,526-14,259 of SEQ ID NO: 105, which correspond to the nucleotide sequence encoding the recombinant viral genome. [78.] An isolated recombinant polynucleotide described in any one of [1] to

[77] , which does not contain a nucleotide sequence encoding an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, or L4 pVIII / hexone-related precursor. A host cell containing an isolated recombinant polynucleotide described in any one of [79.][1]~

[78] . [80.] The host cell according to

[79] , wherein the host cell is a bacterial cell. [81.] The host cell according to

[79] , wherein the host cell is an Escherichia coli (E. coli) cell. [82.] The host cell according to

[79] , wherein the host cell is a eukaryotic cell. [83.] The host cell according to

[79] , wherein the host cell is a mammalian cell. [84.] The host cell according to

[79] , wherein the host cell is HEK293 cell, HEK-derived cell, CHO cell, CHO-derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. A method for producing isolated recombinant polynucleotides according to any one of [1] to

[78] , comprising incubating a host cell according to any one of

[79] to

[84] under suitable conditions. The method according to

[85] , comprising incubating the host cells described in

[86] ,

[80] , or

[81] under suitable conditions. [87.] A method for producing rAAV particles, a) Prepare a cell culture containing cells, b) The cells, i. A polynucleotide described in any one of [1] to

[73] , and ii. A polynucleotide comprising a genome containing at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding the gene product, which is competently bound to a sequence that directs the expression of the gene product in a target cell. Introducing, c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. The method, including the method described above. [88.] A method for producing rAAV particles, a) Prepare a cell culture containing cells, b) Introducing a polynucleotide described in any one of

[74] to

[77] into the cells, c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. The method, including the method described above. [89.] The method according to

[87] or

[88] , wherein the introduction of the polynucleotide(s) into the cell is carried out by transfusion. [90.] The method according to any one of

[87] to

[89] , wherein the cells are mammalian cells. [91.] The method according to any one of

[87] to

[89] , wherein the cells are insect cells. [92.] The method according to any one of

[87] to

[89] , wherein the cells are HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, or PerC6 cells. [93.] The method according to any one of

[87] to

[89] , wherein the cells are HEK293 cells. [94.] The method according to any one of

[87] to

[93] , wherein the cell culture is a suspension culture or an adherent culture. [95.] The method according to any one of

[87] to

[94] , further comprising recovering the rAAV particles. [96.] The method according to any one of

[87] to

[95] , wherein the method produces rAAV particles measured as GC / ml in greater quantities than a reference method using a polynucleotide having a helper function including the nucleotide sequence of SEQ ID NO: 44. [97.] The method according to any one of

[87] to

[95] , wherein the method generates at least about twice as many rAAV particles as measured as GC / ml compared to a reference method using a polynucleotide having a helper function including the nucleotide sequence of SEQ ID NO: 44. [98.] The method according to any one of

[87] to

[95] , wherein the method generates a population of rAAV particles containing complete capsids in greater quantities than a reference method using a polynucleotide containing a helper function including the nucleotide sequence of SEQ ID NO: 44. [99.] The method according to any one of

[87] to

[98] , wherein the cell culture has a volume of approximately 50 liters to approximately 20,000 liters. [100.] The method according to any one of

[87] to

[99] , wherein the gene product is a polypeptide or a double-stranded RNA molecule. [101.] The method according to

[0100] , wherein the gene product is a polypeptide. [102.] The method according to

[0101] , wherein the gene product is anti-VEGF Fab, anti-kallikrein antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1). [103.] The gene products include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinocysin (RS1), muscle Endoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), A Reelsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neuruturin (NRTN), neuruturin The method according to

[0101] , wherein the fusiform protein is rotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial code NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion. [104.] The method according to

[0101] , wherein the gene product is dystrophin or microdystrophin. [105.] The method according to

[0100] , wherein the gene product is a microRNA or antisense RNA.

[0015] Further other features and advantages of the compositions and methods described herein will become clearer from the following detailed description when read in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0016] [Figure 1] Map of pHRC plasmid #1. [Figure 2] Map of pHRC plasmid #2. [Figure 3] Map of pHRC plasmid #3. [Figure 4] Generation of TG-A rAAV8 particles using pHRC#1, pHRC#2, or pHRC#3 plasmid and cis TG-A plasmid. [Figure 5] Generation of TG-A rAAV8 particles using pHRC#3 plasmid and cis TG-A plasmid. [Figure 6] Generation of TG-B rAAV8 particles using pHRC#3 plasmid and cis TG-B plasmid. m1, m2, and m3 represent different absolute masses of pHRC plasmid #3 used. [Figure 7] Map of pHRC plasmid #4. [Figure 8] Map of pHRC plasmid #5. [Figure 9] Generation of TG-A rAAV8 particles using pHRC#3, pHRC#4, or pHRC#5 plasmids and cis TG-A plasmids. [Figure 10] Generation of TG-A rAAV8 particles using pHRC#3 plasmid or pHRC#7 plasmid and cis TG-A plasmid. [Figure 11]Generation of TG-A rAAV8 particles using Helper #5 or Helper #10 plasmid. [Figure 12] Map of pHRC plasmid #7. [Figure 13] Generation of TG-A rAAV8 particles using pHRC#3, pHRC#5, or pHRC#7 plasmids. [Figure 14A] Generation of TG-A rAAV8 particles using different expression systems on days 1, 2, and 3 after transfusion. ddPCR titers are shown. [Figure 14B] Generation of TG-A rAAV8 particles using different expression systems on days 1, 2, and 3 after transfusion. ELISA titers are shown. [Figure 14C] Generation of TG-A rAAV8 particles using different expression systems on days 1, 2, and 3 after transfusion. Full percentage calculations based on ddPCR and ELISA titers are shown. [Figure 15A] Western blot analysis for detection of viral capsid proteins within TG-A rAAV8 particles. Western blot analysis of VP1, VP2, and VP3 capsid proteins. [Figure 15B] Western blot analysis for detection of viral capsid proteins within TG-A rAAV8 particles. Comparison of the change in total VP1 / 2 / 3 in two cell lines using different plasmids. [Figure 15C] Western blot analysis for detection of viral capsid proteins within TG-A rAAV8 particles. Comparison of the change in total VP1 / 2 / 3 in two cell lines using different plasmids. [Figure 15D] Western blot analysis for detection of viral capsid proteins within TG-A rAAV8 particles. Changes in VP1 / 2 / 3 in cell line clone 4-11A4 (normalized to vinculin). [Figure 16A] Western blot analysis for detection of replicase proteins in TG-A rAAV8 particles. Western blot analysis of Rep78, Rep52, and Rep40 proteins. [Figure 16B] Western blot analysis to detect replicase proteins within TG-A rAAV8 particles. Comparison of the change in total Rep78 / Rep52 / Rep40 in two cell lines using different plasmids. [Figure 16C] Western blot analysis for detection of replicase proteins in TG-A rAAV8 particles. Change ratios of Rep78 / Rep52 / Rep40 in cell line clone 1 (normalized to vinculin). [Figure 16D] Western blot analysis for detection of replicase proteins in TG-A rAAV8 particles. Changes in Rep78 / Rep52 / Rep40 in cell line clone 4-11A4 (normalized to vinculin). [Figure 17] Generation of TG-B rAAV8 particles using the original helper, helper #3, helper #5, pHRC#3, pHRC#5, or pHRC#7 plasmid. [Figure 18] Generation of TG-C rAAV9 particles using the original helper, helper #3, helper #5, or pHRC#8 plasmid. [Figure 19] Generation of TG-D rAAV9 particles using the original helper, helper #3, helper #5, or pHRC#8 plasmid. [Figure 20] Generation of TG-A rAAV9 particles using the original helper, helper #3, helper #5, pHRC#8, pHRC#11, or pHRC#14 plasmid. [Figure 21] Generation of TG-D rAAV9 particles using the original helper, helper #3, helper #5, pHRC#8, pHRC#11, or pHRC#14 plasmid. [Figure 22] Generation of TG-A rAAV2 particles using the original helper, helper #3, helper #5, pHRC#9, pHRC#12, or pHRC#15 plasmid. [Figure 23]Generation of TG-A rAAV8 particles using Helper #5, pHRC#5, or pHRC#7 plasmid. A: ddPCR titer, B: Full percentage calculation based on analytical ultracentrifugation (AUC). [Figure 24] Map of pHRC plasmid #35. [Figure 25] Map of pHRC plasmid #36. [Figure 26] Map of pHRC plasmid #37. [Figure 27] Map of pHRCG plasmid #1. [Figure 28] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC#7, or pHRCG#1 plasmid. [Figure 29] Map of pHRCG plasmid #2. [Figure 30] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC #7, pHRCG #1, or pHRCG #2 plasmid. [Figure 31] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC #7, or pHRCG #1 plasmid. A: Experiment 1, B: Experiment 2. [Figure 32] Map of pHRCG plasmid #3. [Figure 33] Map of pHRCG plasmid #4. [Figure 34A] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC #7, pHRCG #1, pHRCG #3, or pHRCG #4 plasmid. ddPCR titers are shown. [Figure 34B] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC #7, pHRCG #1, pHRCG #3, or pHRCG #4 plasmid. ELISA titers are shown. [Figure 34C]Generation of TG-A rAAV8 particles using the original helper, helper #5, pHRC #7, pHRCG #1, pHRCG #3, or pHRCG #4 plasmid. Full percentage calculations based on ddPCR and ELISA titers are shown. [Figure 35A] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC#3, pHRC#5, pHRC#7, or pHRCG#1 plasmid. ddPCR titers are shown. [Figure 35B] Generation of TG-A rAAV8 particles using the original helper plasmid, helper #5, pHRC#3, pHRC#5, pHRC#7, or pHRCG#1 plasmid. ELISA titer is shown. [Figure 35C] Generation of TG-A rAAV8 particles using the original helper, helper #5, pHRC#3, pHRC#5, pHRC#7, or pHRCG#1 plasmid. Full percentage calculations based on ddPCR and ELISA titers are shown. [Figure 36] Generation of TG-A rAAV2 particles using the original helper, helper #3, helper #5, pHRC#15, pHRC#17, pHRC#18, pHRC#19, pHRC#20, pHRC#21, or pHRCG#22 plasmid. [Figure 37A] The mAAP mutation affects rAAV8 virus generation. Generation of TG-A(A), TG-B(B), and TG-D(C)rAAV8 particles using the original helper, helper #3, helper #5, pHRC#7, pHRC#23, pHRC#24, pHRC#25, pHRC#26, pHRC#27, or pHRCG#28 plasmids. [Figure 37B] Refer to the explanation in Figure 37A. [Figure 37C] Refer to the explanation in Figure 37A. [Figure 38]The mAAP mutation affects rAAV9 virus generation. Generation of TG-A rAAV9 particles using the original helper, helper #3, helper #5, pHRC#8, pHRC#29, pHRC#30, pHRC#31, pHRC#32, pHRC#33, or pHRCG#34 plasmid. [Figure 39] The mAAP mutation affects rAAV9 virus generation. Generation of TG-D rAAV9 particles using the original helper, helper #3, helper #5, pHRC#8, pHRC#29, pHRC#30, pHRC#31, pHRC#32, pHRC#33, or pHRCG#34 plasmid. [Figure 40A] The mAAP mutation affects rAAV8 virus secretion. Generation of TG-A(A), TG-B(B), and TG-D(C)rAAV8 particles using the original helper, helper #3, helper #5, pHRC#7, pHRC#23, pHRC#24, pHRC#25, pHRC#26, pHRC#27, or pHRCG#28 plasmids. [Figure 40B] Refer to the explanation in Figure 40A. [Figure 40C] Refer to the explanation in Figure 40A. [Figure 41A] Full percentage of TG-A(A), TG-B(B), and TG-D(C)rAAV8 particles generated using the original helper, helper #3, helper #5, pHRC#7, pHRC#23, pHRC#24, pHRC#25, pHRC#26, pHRC#27, or pHRCG#28 plasmid. Full percentage calculation is based on ddPCR and ELISA titers. [Figure 41B] Refer to the explanation in Figure 41A. [Figure 41C] Refer to the explanation in Figure 41A. [Figure 42] Full percentage of TG-A(A) and TG-D(B)rAAV9 particles generated using the original helper, helper #3, helper #5, pHRC#8, pHRC#29, pHRC#30, pHRC#31, pHRC#32, pHRC#33, or pHRCG#34 plasmid. Full percentage calculation is based on ddPCR and ELISA titers. [Modes for carrying out the invention]

[0017] Detailed explanation In one embodiment, improved recombinant polynucleotides and plasmids encoding helper functions, AAV rep genes, and AAV cap genes are provided, suitable for use in the production of recombinant AAV particles. In some embodiments, the helper function comprises a nucleotide sequence encoding an adenovirus E2A DNA-binding protein, a nucleotide sequence encoding an adenovirus E4 polypeptide, and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the polynucleotides and plasmids do not contain nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursors. In some embodiments, the polynucleotides and plasmids are smaller than previously available polynucleotides and plasmids encoding helper functions and AAV rep / cap genes, suitable for use in the production of recombinant AAV particles. In some embodiments, using the improved polynucleotides and plasmids described herein for the production of recombinant AAV particles results in increased rAAV yield.

[0018] In one embodiment, improved recombinant polynucleotides and plasmids are provided herein that encode a helper function, an AAV rep gene, an AAV cap gene, and a recombinant AAV viral genome suitable for use in the generation of recombinant AAV particles. In some embodiments, the recombinant AAV viral genome comprises at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product, responsively bound to a sequence that directs the expression of the gene product in a target cell. In some embodiments, the helper function comprises a nucleotide sequence encoding an adenovirus E2A DNA-binding protein, a nucleotide sequence encoding an adenovirus E4 polypeptide, and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the polynucleotides and plasmids do not contain nucleotide sequences encoding an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor.

[0019] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure relates. To facilitate understanding of the methods of this disclosure, several terms and expressions are defined below.

[0020] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its modifiers, derivatives, or pseudotypes. Unless otherwise required, the term encompasses all subtypes, as well as both the natural and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as their modifiers, derivatives, or pseudotypes. "Primate AAV" refers to AAVs that infect primates, "non-primate AAV" refers to AAVs that infect non-primate mammals, and "bovine AAV" refers to AAVs that infect bovine mammals, and so on.

[0021] The term "recombination" as applied to AAV particles means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is essentially different from the AAV particle itself.

[0022] Recombinant adeno-associated virus particles, or "rAAV particles," refer to viral particles composed of at least one AAV capsid protein and a capsidized polynucleotide rAAV vector genome containing heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, e.g., a transgene to be delivered to mammalian cells). rAAV particles can be any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or their derivatives / modifiers / pseudotypes), including any modifiers, derivatives, or pseudotypes. Such AAV serotypes and derivatives / modifiers / pseudotypes, as well as methods for generating such serotypes / derivatives / modifiers / pseudotypes, are known in the art (see, for example, Asokan et al., Mol.Ther. 20(4):699-708 (2012)).

[0023] The rAAV particles of this disclosure may be any serotype or any combination of serotypes (for example, a population of rAAV particles containing two or more serotypes (for example, two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or two or more combinations thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles.

[0024] In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or derivatives, modifiers, or pseudotypes thereof. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype consisting of AAV8, AAV9, or derivatives, modifiers, or pseudotypes thereof.

[0025] The term “cell culture” refers to cells grown attached to or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., as well as the components of the supernatant or suspension itself, including, but not limited to, rAAV particles, cells, cell debris, cell contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, lipids, and flocculants. Large-scale approaches such as bioreactors (including suspension cultures and adherent cells grown attached to microcarriers or macrocarriers in agitated bioreactors) are also encompassed by the term “cell culture.” Cell culture procedures for both large-scale and small-scale protein production are also included in this disclosure. In some embodiments, the term “cell culture” refers to cells grown in suspension. In some embodiments, the term “cell culture” refers to adherent cells grown attached to microcarriers or macrocarriers in agitated bioreactors. In some embodiments, the term “cell culture” refers to cells grown in perfusion culture. In some embodiments, the term “cell culture” refers to cells grown in an alternating tangential flow (ATF) supported high-density perfusion culture.

[0026] As used herein, the terms “purify,” “separate,” “isolate,” “isolate,” “remove,” or “isolate” mean increasing the degree of purity of a target product (e.g., rAAV particles and rAAV genome) from a sample containing the target product and one or more impurities. Typically, the degree of purity of a target product is increased by removing (completely or incompletely) at least one impurity from the sample. In some embodiments, the degree of purity of rAAV in a sample is increased by removing (completely or incompletely) one or more impurities from the sample using the methods described herein.

[0027] For example, modifying the amount of an ingredient in a composition, the concentration of an ingredient in a composition, the flow rate, the rAAV particle yield, the feed volume, the salt concentration, similar values, and ranges thereof, “about” as used in the methods provided herein means variations in numerical quantities that may result from, for example, typical measurement and handling procedures used to prepare a concentrate or working solution; accidental errors in these procedures; differences in the manufacture, source, or purity of the ingredients used in preparing the composition or performing the method; and similar considerations. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration ages. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration is mixed or processed. Whether modified by the term “about,” a claim includes an equivalent of that quantity. In some embodiments, the term “about” means a range of about 10 to 20 percent more or less than the number or range indicated. In further embodiments, “about” means plus or minus 10 percent of the number or range indicated. For example, “about 10%” refers to a range of 9% to 11%.

[0028] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0029] Whenever embodiments described herein using the phrase "including" should be understood as also providing other similar embodiments described in terms of "consisting of" and / or "essentially consisting of".

[0030] When the term "and / or" is used herein in an expression such as "A and / or B," it is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, when the term "and / or" is used in an expression such as "A, B, and / or C," it is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] Where embodiments of the Disclosure describe a Markush group or other alternative groups, the Method of the Disclosure encompasses not only the entire group as a whole, but also each individual member of the group, all possible subgroups of a principal group, and even principal groups in which one or more group members are absent. The Method of the Disclosure also anticipates the express exclusion of one or more of the group members in the Method of the Disclosure.

[0032] Recombinant polynucleotides In some embodiments, the Disclosure provides isolated recombinant polynucleotides encoding one or more helper functions and AAV rep and AAV cap genes, which can facilitate the generation of recombinant AAV particles in host cells (e.g., HEK cells). In some embodiments, the isolated recombinant polynucleotides described herein include (a) a nucleotide sequence encoding adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, and (d) nucleotide sequences encoding AAV rep and AAV cap genes. In some embodiments, the adenovirus E4 polypeptide includes E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is immediately upstream of the nucleotide sequence encoding the AAV rep gene. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is separated from the nucleotide sequence encoding the AAV rep gene by about 1 to about 10,000 nucleotides. In some embodiments, the isolated recombinant polynucleotide does not contain nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor.In some embodiments, the nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor are the corresponding nucleotide sequences of pAdDeltaF6. In some embodiments, the nucleotide sequences encoding a protein or polypeptide (e.g., E2A DBP, E4 polypeptide, AAV rep, and AAV cap) or RNA (e.g., VA RNA I) include a promoter responsively bound to the coding region of the protein or polypeptide, or to the nucleotide sequence containing RNA. In some embodiments, the nucleotide sequences encoding a protein or polypeptide include a promoter responsively bound to the nucleotide sequence containing the coding region and a poly(A) signal.

[0033] In some embodiments, the isolated recombinant polynucleotide described herein comprises a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 polypeptide, and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the isolated recombinant polynucleotide described herein comprises a nucleotide sequence encoding adenovirus E2A DBP and a nucleotide sequence encoding adenovirus E4 polypeptide. In some embodiments, the isolated recombinant polynucleotide described herein comprises a nucleotide sequence encoding adenovirus E2A DBP and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the isolated recombinant polynucleotide described herein comprises a nucleotide sequence encoding adenovirus E4 polypeptide and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the isolated recombinant polynucleotide described herein comprises a nucleotide sequence encoding adenovirus E2A DBP. In some embodiments, the isolated recombinant polynucleotide described herein comprises a nucleotide sequence encoding adenovirus E4 polypeptide. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the isolated recombinant polynucleotides described herein include a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the isolated recombinant polynucleotides do not include a nucleotide sequence encoding adenovirus ITR, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor.In some embodiments, the nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor are the corresponding nucleotide sequences of pAdDeltaF6. In some embodiments of the isolated recombinant polynucleotide, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequence encoding adenovirus E4 polypeptide are oriented in opposite 5'→3' directions. In some embodiments of the isolated recombinant polynucleotide, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequence encoding adenovirus E4 polypeptide are oriented in the same 5'→3' direction. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequences encoding the AAV rep gene and AAV cap gene are oriented in opposite 5'→3' directions.

[0034] In some embodiments, the isolated recombinant polynucleotide described herein comprises (a) a nucleotide sequence encoding adenovirus E2A DBP, (b) a nucleotide sequence encoding adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, and (d) nucleotide sequences encoding the AAV rep gene and the AAV cap gene, wherein the 5'→3' order of the nucleotide sequences is (d)-(a)-(b)-(c). In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequence encoding adenovirus E4 polypeptide are in opposite 5'→3' directions. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene are in opposite 5'→3' directions. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequence encoding adenovirus E4 polypeptide are oriented in opposite 5'→3' directions, and the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequences encoding the AAV rep gene and AAV cap gene are oriented in opposite 5'→3' directions. In some embodiments, the isolated recombinant polynucleotide does not contain the nucleotide sequence encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-associated precursor. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II.

[0035] In some embodiments, the Disclosure provides isolated recombinant polynucleotides encoding one or more helper functions, an AAV rep gene, an AAV cap gene, and a recombinant AAV viral genome, the polynucleotides capable of facilitating the generation of recombinant AAV particles within a host cell (e.g., a HEK cell). In some embodiments, the isolated recombinant polynucleotides described herein include: (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP); (b) a nucleotide sequence encoding an adenovirus E4 polypeptide; (c) a nucleotide sequence encoding an adenovirus VA RNA I; (d) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene; and (e) a nucleotide sequence encoding a recombinant viral genome comprising at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product, responsively bound to a sequence directing the expression of the gene product in a target cell. In some embodiments, the adenovirus E4 polypeptide includes E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequence encoding the AAV rep gene and AAV cap gene comprises a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequence encoding the AAV rep gene and AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is immediately upstream of the nucleotide sequence encoding the AAV rep gene. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is separated from the nucleotide sequence encoding the AAV rep gene by about 1 to about 10,000 nucleotides.In some embodiments, the isolated recombinant polynucleotides do not contain nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursors. In some embodiments, the nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursors are the corresponding nucleotide sequences of pAdDeltaF6. In some embodiments, nucleotide sequences encoding proteins or polypeptides (e.g., E2A DBP, E4 polypeptide, AAV rep, and AAV cap) or RNA (e.g., VA RNA I) include a promoter responsively bound to the coding region of the protein or polypeptide, or to the nucleotide sequence containing RNA. In some embodiments, nucleotide sequences encoding proteins or polypeptides include a promoter responsively bound to the nucleotide sequence containing the coding region and a polyA signal. In some embodiments, the recombinant polynucleotides described herein are sufficient on their own to promote the generation of recombinant AAV particles within host cells (e.g., HEK cells).

[0036] In some embodiments, the isolated recombinant polynucleotide described herein comprises (a) a nucleotide sequence encoding adenovirus E2A DBP, (b) a nucleotide sequence encoding adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, (d) a nucleotide sequence encoding the AAV rep gene and the AAV cap gene, and (e) a nucleotide sequence encoding the recombinant AAV virus genome, wherein the 5'→3' order of the nucleotide sequences is (d)-(a)-(b)-(e)-(c). In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequence encoding adenovirus E4 polypeptide are in opposite 5'→3' directions. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene are in opposite 5'→3' directions. In some embodiments, the nucleotide sequences encoding the AAV rep gene and AAV cap gene and the nucleic acid sequences encoding the gene products are oriented in opposite 5'→3' directions. In some embodiments, (i) the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequence encoding adenovirus E4 polypeptide are oriented in opposite 5'→3' directions, (ii) the nucleotide sequence encoding adenovirus E2A DBP and the nucleotide sequences encoding the AAV rep gene and AAV cap gene are oriented in opposite 5'→3' directions, and (iii) the nucleotide sequences encoding the AAV rep gene and AAV cap gene and the nucleic acid sequences encoding the gene products are oriented in opposite 5'→3' directions. In some embodiments, the isolated recombinant polynucleotide does not contain nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursors.In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I also encodes adenovirus VA RNA I and VA RNA II.

[0037] Adenovirus E2A DNA-binding protein In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized nucleotide sequence. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 60. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 60. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes SEQ ID NO: 60. In some embodiments, the adenovirus E2A DBP polypeptide includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide includes an amino acid sequence having at least 90% identity with SEQ ID NO: 45.In some embodiments, the adenovirus E2A DBP polypeptide contains an amino acid sequence having at least 95% identity to SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide contains an amino acid sequence having at least 98% identity to SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide contains the amino acid sequence of SEQ ID NO: 45. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is adsorbed to a promoter and a poly(A) signaling molecule.

[0038] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide includes an amino acid sequence having at least 90% identity with SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide includes an amino acid sequence having at least 95% identity with SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide includes an amino acid sequence having at least 98% identity with SEQ ID NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide includes the amino acid sequence of SEQ ID NO: 45. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is admissibly bound to a promoter and a poly(A) signal.

[0039] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is responsively bound to the adenovirus E2A promoter. In some embodiments, the adenovirus E2A promoter includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2. In some embodiments, the adenovirus E2A promoter includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 2. In some embodiments, the adenovirus E2A promoter includes the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is responsively bound to a promoter that is not the adenovirus E2A promoter.

[0040] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, which is optionally conjugated to the adenovirus E2A promoter and the polyA signaling molecule, encompasses the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, the nucleotide sequence encoding adenovirus E2A DBP, and optionally the polyA signaling molecule, in the 3'→5' direction. In some embodiments, the relative direction of the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, the nucleotide sequence encoding adenovirus E2A DBP, and optionally the polyA signaling molecule is the same as that of pAdDeltaF6. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, which is responsively bound to the adenovirus E2A promoter, includes the nucleotide sequence of Sequence ID No. 3.In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter and polyA signaling molecule, includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter and polyA signaling molecule, includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter and polyA signaling molecule, includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter and polyA signaling molecule, includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, which is responsively bound to the adenovirus E2A promoter and polyA signaling pathway, includes the nucleotide sequence of Sequence ID No. 4.

[0041] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, encompasses, in the 3'→5' direction, the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexone assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP, wherein the adenovirus L4 100k / hexone assembly gene contains an N-terminal deletion of the L4 100k / hexone assembly polypeptide. In some embodiments, the N-terminal deletion does not affect the L4 100k / hexone assembly promoter. In some embodiments, the N-terminal deletion corresponds to the sequence of SEQ ID NO: 21. In some embodiments, the relative orientation of the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP is the same as that of pAdDeltaF6. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 22.In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, which is operablely bound to the adenovirus E2A promoter, includes the nucleotide sequence of Sequence ID No. 22. In some embodiments, the nucleotide sequences encoding adenovirus E2A DBP and the adenovirus E2A promoter further include an operablely bound poly(A) signal.

[0042] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, includes the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP in the 3'→5' direction, where the adenovirus L4 100k / hexon assembly gene contains a mutation in the start codon of the L4 100k / hexon assembly polypeptide. In some embodiments, the relative orientation of the nucleotide sequences encoding the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, and adenovirus E2A DBP is the same as that of pAdDeltaF6. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, which is responsively bound to the adenovirus E2A promoter, includes the nucleotide sequence of SEQ ID NO: 23.In some embodiments, the nucleotide sequences encoding adenovirus E2A DBP and adenovirus E2A promoter further include an actionably bound poly(A) signal.

[0043] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, encompasses the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP in the 3'→5' direction, where the adenovirus L4 22K / 33K gene contains a mutation in the start codon of the L4 22K / 33K polypeptide. In some embodiments, the relative orientation of the nucleotide sequences encoding the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, and adenovirus E2A DBP is the same as that of pAdDeltaF6. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 24. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 24. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 24. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, operably bound to the adenovirus E2A promoter, includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 24. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, which is actionably bound to the adenovirus E2A promoter, includes the nucleotide sequence of SEQ ID NO: 24.In some embodiments, the nucleotide sequences encoding adenovirus E2A DBP and adenovirus E2A promoter further include an actionably bound poly(A) signal.

[0044] In some embodiments, a nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, encompasses, in the 3'→5' direction, the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexone assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP, wherein the L4 100k / hexone assembly gene contains an N-terminal deletion of the L4 100k / hexone assembly polypeptide that includes the start codon of the L4 100k / hexone assembly polypeptide but does not include the start codon of the L4 22K / 33K polypeptide. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, encompasses, in the 3'→5' direction, the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexone assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP, wherein the L4 100k / hexone assembly gene contains an N-terminal deletion of the L4 100k / hexone assembly polypeptide, and all or part of the L4 100k / hexone assembly polypeptide is deleted without disruption of the L4 22K / 33K start codon. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, includes, in the 3'→5' direction, the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexone assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP, wherein the L4 100k / hexone assembly gene includes an N-terminal deletion of the L4 100k / hexone assembly polypeptide that includes the start codon of the L4 100k / hexone assembly polypeptide but does not include the L4 22K / 33K promoter.In some embodiments, a nucleotide sequence encoding adenovirus E2A DBP, responsively bound to the adenovirus E2A promoter, encompasses, in the 3'→5' direction, the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexone assembly gene, and the nucleotide sequence encoding adenovirus E2A DBP, where the L4 100k / hexone assembly gene includes an N-terminal deletion of the L4 100k / hexone assembly polypeptide, starting at the L4 100k / hexone assembly polypeptide start codon and ending immediately adjacent to the L4 22K / 33K polypeptide. In some embodiments, the relative orientation of the nucleotide sequences encoding the adenovirus E2A promoter, the adenovirus L4 22K / 33K gene, the adenovirus L4 100k / hexon assembly gene, and the adenovirus E2A DBP is the same as that of pAdDeltaF6. In some embodiments, the nucleotide sequences encoding the adenovirus E2A DBP and the adenovirus E2A promoter further include an actionably bound poly(A) signal.

[0045] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is responsively bound to the CMV early promoter. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is responsively bound to an engineered CMV early promoter, or a transcriptionally active fragment or portion thereof. In some embodiments, the CMV early promoter includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 121. In some embodiments, the CMV early promoter includes the nucleotide sequence of SEQ ID NO: 121. Engineered CMV early promoters or their transcriptionally active fragments or portions are known to those skilled in the art, for example, as disclosed in International Application No. PCT / US2023 / 061014 (filed January 20, 2023) (the whole of which is incorporated herein by reference).

[0046] In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP is adsorbed to an inducible promoter.

[0047] Adenovirus E4 polypeptide In some embodiments, the nucleotide sequence encoding the adenovirus E4 polypeptide encodes an E4 polypeptide containing E4 ORF6. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide contains a nucleotide sequence having at least 90% identity with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide contains a nucleotide sequence having at least 95% identity with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide contains a nucleotide sequence having at least 98% identity with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide contains SEQ ID NO: 6. In some embodiments, the adenovirus E4 ORF6 polypeptide contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide contains an amino acid sequence having at least 90% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide contains an amino acid sequence having at least 95% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide contains an amino acid sequence having at least 98% identity with SEQ ID NO: 46. In some embodiments, the E4 ORF6 polypeptide contains the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding adenovirus E4 ORF6 is adsorbed to a promoter and a poly(A) signal.

[0048] In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide includes a nucleotide sequence encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide includes an amino acid sequence having at least 90% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide includes an amino acid sequence having at least 95% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide includes an amino acid sequence having at least 98% identity with SEQ ID NO: 46. In some embodiments, the E4 ORF6 polypeptide includes the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding adenovirus E4 ORF6 is adsorbably bound to a promoter and a poly(A) signal.

[0049] In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide is responsively bound to the adenovirus E4 promoter. In some embodiments, the adenovirus E4 promoter includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 5. In some embodiments, the adenovirus E4 promoter includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 5. In some embodiments, the adenovirus E4 promoter includes the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 polypeptide is responsively bound to a promoter other than the adenovirus E4 promoter.

[0050] In some embodiments, the nucleotide sequence encoding the adenovirus E4 polypeptide encodes the E4 polypeptide comprising E4 ORF6 and ORF7. E4 ORF7 is also referred to in the art as E4 ORF6 / 7. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a codon-optimized nucleotide sequence. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptides includes SEQ ID NO: 61.Those skilled in the art will understand that the polynucleotide of SEQ ID NO: 8 encodes two alternating splicing mRNAs, one each of ORF6 and ORF7 (i.e., ORF6 / 7). The ORF6 mRNA contains nucleotide sequences corresponding to residues 1-885 of SEQ ID NO: 8. The ORF7 (i.e., ORF6 / 7) mRNA contains nucleotide sequences corresponding to residues 1-174 (exon 1) and 886-1164 (exon 2) of SEQ ID NO: 8. In some embodiments, the adenovirus E4 ORF6 and ORF7 polypeptides contain amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 46 and 120, respectively. In some embodiments, the adenovirus E4 ORF6 polypeptide contains amino acid sequences having at least 90% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide contains an amino acid sequence having at least 95% identity to SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide contains an amino acid sequence having at least 98% identity to SEQ ID NO: 46. In some embodiments, the E4 ORF6 polypeptide contains the amino acid sequence of SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF7 polypeptide contains an amino acid sequence having at least 90% identity to SEQ ID NO: 120. In some embodiments, the adenovirus E4 ORF7 polypeptide contains an amino acid sequence having at least 95% identity to SEQ ID NO: 120. In some embodiments, the adenovirus E4 ORF7 polypeptide contains an amino acid sequence having at least 98% identity to SEQ ID NO: 120. In some embodiments, the E4 ORF7 polypeptide contains the amino acid sequence of SEQ ID NO: 120. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides are responsively bound to a promoter and a poly(A) signal.

[0051] In some embodiments, the nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides include a nucleotide sequence encoding an ORF6 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 46, and an ORF7 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 120. In some embodiments, the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 46. In some embodiments, the E4 ORF6 polypeptide contains the amino acid sequence of SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF7 polypeptide contains an amino acid sequence having at least 90% identity to SEQ ID NO: 120. In some embodiments, the adenovirus E4 ORF7 polypeptide contains an amino acid sequence having at least 95% identity to SEQ ID NO: 120. In some embodiments, the adenovirus E4 ORF7 polypeptide contains an amino acid sequence having at least 98% identity to SEQ ID NO: 120. In some embodiments, the E4 ORF7 polypeptide contains the amino acid sequence of SEQ ID NO: 120. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides are adsorbably bound to a promoter and a poly(A) signal.

[0052] In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides are responsively bound to the adenovirus E4 promoter. In some embodiments, the adenovirus E4 promoter includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 5. In some embodiments, the adenovirus E4 promoter includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 5. In some embodiments, the adenovirus E4 promoter includes the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides are responsively bound to a promoter other than the adenovirus E4 promoter.

[0053] In some embodiments, the nucleotide sequence encoding the adenovirus polypeptide is responsively bound to the CMV initial promoter. In some embodiments, the nucleotide sequence encoding the adenovirus E4 polypeptide is responsively bound to the manipulated CMV initial promoter, or a transcriptionally active fragment or portion thereof. In some embodiments, the CMV initial promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 121. In some embodiments, the CMV initial promoter comprises the nucleotide sequence of SEQ ID NO: 121. The manipulated CMV initial promoter or its transcriptionally active fragment or portion thereof is known to those skilled in the art, for example, as disclosed in International Application No. PCT / US2023 / 061014 (filed January 20, 2023) (the whole thereof is incorporated herein by reference). In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6.

[0054] In some embodiments, the nucleotide sequence encoding the adenovirus E4 polypeptide is adsorbed to an inducible promoter. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7, or E4 ORF6.

[0055] Adenovirus VARNA In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 54. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 54. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 54. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 54. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I includes SEQ ID NO: 54.

[0056] In some embodiments, the nucleotide sequence encoding adenovirus VA RNA II includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA II includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA II includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA II includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA II includes SEQ ID NO: 55.

[0057] In some embodiments, the nucleotide sequences encoding adenovirus VA RNA I and VA RNA II include nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequences encoding adenovirus VA RNA I and VA RNA II include nucleotide sequences having at least 90% identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequences encoding adenovirus VA RNA I and VA RNA II include nucleotide sequences having at least 95% identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequences encoding adenovirus VA RNA I and VA RNA II include nucleotide sequences having at least 98% identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequences encoding adenovirus VA RNA I and VA RNA II include SEQ ID NO: 9.

[0058] Polynucleotides encoding E2A DBP, E4 polypeptide, and VA RNA In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment containing a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 polypeptide, and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the promoter expressing adenovirus E2A DBP and the promoter expressing adenovirus E4 polypeptide are the same. In some embodiments, the promoter expressing adenovirus E2A DBP and the promoter expressing adenovirus E4 polypeptide are different.

[0059] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 10. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 10. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 10. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 10. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0060] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 11. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 11. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0061] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 56. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 56. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 56. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 56. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0062] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 57. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 57. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 57. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 57. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 57. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0063] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 25. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 25. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 25. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 25. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0064] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 26. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 26. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 26. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 26. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0065] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 27. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 27. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 27. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 27. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0066] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 28. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 28. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 28. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 28. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0067] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 29. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 29. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 29. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 29. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0068] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 30. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 30. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 30. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 30. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0069] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 31. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 31. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 31. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0070] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 32. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 32. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0071] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 33. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 33. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 33. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 33. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0072] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 34. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 34. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 34. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 34. In some embodiments, the linker includes the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0073] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides, and a nucleotide sequence encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 106. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 106. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 106. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 106. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding regions include the nucleotide sequence of SEQ ID NO: 61.

[0074] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides, and a nucleotide sequence encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 107. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 107. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 107. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 107. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding regions include the nucleotide sequence of SEQ ID NO: 61.

[0075] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 108. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 108. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 108. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 108. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0076] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 109. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 109. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 109. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 109. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 109. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0077] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 56 or 57. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 56 or 57. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 56 or 57. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 56 or 57. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 56 or 57. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0078] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides, and a nucleotide sequence encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 106 or 107. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 106 or 107. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 106 or 107. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 106 or 107. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 106 or 107. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0079] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides, and a nucleotide sequence encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108 or 109. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 108 or 109. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 108 or 109. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 108 or 109. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 108 or 109. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0080] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides, and a nucleotide sequence encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the plasmid includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 122-130 or 131. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity with SEQ ID NOs. 122-130 or 131. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity with SEQ ID NOs. 122-130 or 131. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity with SEQ ID NOs. 122-130 or 131. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NOs. 122-130 or 131. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO. 60.

[0081] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity with SEQ ID NOs: 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NOs: 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NOs. 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260. In some embodiments, the fragment comprises a nucleotide sequence of SEQ ID NOs. 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260.In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0082] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides, and a nucleotide sequence encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 145 or 146. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 145 or 146. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 145 or 146. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 145 or 146. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 145 or 146. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0083] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 156, 157, or 158. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity with SEQ ID NOs. 156, 157, or 158. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity with SEQ ID NOs. 156, 157, or 158. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity with SEQ ID NOs. 156, 157, or 158. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 156, 157, or 158. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of SEQ ID NO: 60.

[0084] Polynucleotides encoding E2A DBP and E4 polypeptides In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment containing a nucleotide sequence encoding adenovirus E2A DBP and a nucleotide sequence encoding adenovirus E4 polypeptide. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the promoter expressing adenovirus E2A DBP and the promoter expressing adenovirus E4 polypeptide are the same. In some embodiments, the promoter expressing adenovirus E2A DBP and the promoter expressing adenovirus E4 polypeptide are different.

[0085] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 110. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 110. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 110. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 110. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 110. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0086] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 111. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 111. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 111. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 111. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 111. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0087] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 112. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 112. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 112. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 112. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 112. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0088] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 113. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 113. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 113. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 113. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0089] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 114. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 114. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 114. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 114. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0090] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 115. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 115. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 115. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 115. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 115. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequences encoding the adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding region includes the nucleotide sequence of SEQ ID NO: 61.

[0091] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and a nucleotide sequence encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 116. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 116. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 116. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 116. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides include a codon-optimized coding region. In some embodiments, the codon-optimized E4 ORF6 and ORF7 polypeptide coding regions include the nucleotide sequence of SEQ ID NO: 61.

[0092] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 117. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 117. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 117. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 117. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 117. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60.

[0093] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 277. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 277. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 277. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 277. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 277. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60.

[0094] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 278. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 278. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 278. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 278. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 278. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60.

[0095] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 279. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 279. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 279. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 279. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 279. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP comprises a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region comprises the nucleotide sequence of SEQ ID NO: 60.

[0096] In some embodiments, the isolated recombinant polynucleotide described herein comprises a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP and a nucleotide sequence encoding adenovirus E4 ORF6 polypeptide. In some embodiments, the fragment comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 280, 281, or 282. In some embodiments, the fragment comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 280, 281, or 282. In some embodiments, the fragment comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 280, 281, or 282. In some embodiments, the fragment comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 280, 281, or 282. In some embodiments, the fragment comprises the nucleotide sequence of SEQ ID NO: 280, 281, or 282. In some embodiments, the nucleotide sequence encoding adenovirus E2A DBP includes a codon-optimized coding region. In some embodiments, the codon-optimized E2A DBP coding region includes the nucleotide sequence of sequence number 60.

[0097] AAV rep gene and AAV cap gene In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene, wherein the parvovirus p5 promoter is actionably bound to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the p5 promoter is located about 10 to about 10,000 nucleotides upstream from the start codon of the AAV rep gene.

[0098] In some embodiments, the p5 promoter is located about 5,000 to 10,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to 5,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to 4,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to 3,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 2,000 to 5,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 2,000 to 4,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 2,000 to 3,000 nucleotides upstream from the start codon of the AAV rep.

[0099] In some embodiments, the nucleotide sequences encoding E2A DBP, E4 polypeptide, and / or VA RNA are located between the p5 promoter upstream of the AAV rep start codon and the AAV rep start codon. In some embodiments, the nucleotide sequences encoding E2A DBP, E4 polypeptide, and VA RNA are located between the p5 promoter upstream of the AAV rep start codon and the AAV rep start codon. In some embodiments, the nucleotide sequences encoding E2A DBP, E4 polypeptide, or VA RNA are not located between the p5 promoter upstream of the AAV rep start codon and the AAV rep start codon.

[0100] In some embodiments, the parvovirus p5 promoter is the AAV p5 promoter. In some embodiments, the AAV p5 promoter includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 62. In some embodiments, the AAV p5 promoter includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 62. In some embodiments, the AAV p5 promoter includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 62. In some embodiments, the AAV p5 promoter includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 62. In some embodiments, the AAV p5 promoter includes the nucleotide sequence of SEQ ID NO: 62.

[0101] In some embodiments, the nucleotide sequence encodes the AAV rep gene and the AAV cap gene, and the AAV rep gene and the AAV cap gene have the same serotype. In some embodiments, the AAV rep gene and the AAV cap gene have different serotypes. In some embodiments, the nucleotide sequence encodes the AAV2 rep gene and the AAV2 cap gene. In some embodiments, the nucleotide sequence encodes the AAV2 rep gene and the AAV6 cap gene. In some embodiments, the nucleotide sequence encodes the AAV2 rep gene and the AAV8 cap gene. In some embodiments, the nucleotide sequence encodes the AAV2 rep gene and the AAV9 cap gene.

[0102] In some embodiments, the nucleotide sequence encodes an AAV2 rep gene. In some embodiments, the AAV2 rep gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 63.

[0103] In some embodiments, the AAV2 rep gene contains a nucleotide sequence having at least 90% identity with SEQ ID NO: 63. In some embodiments, the AAV2 rep gene contains a nucleotide sequence having at least 95% identity with SEQ ID NO: 63. In some embodiments, the AAV2 rep gene contains a nucleotide sequence having at least 98% identity with SEQ ID NO: 63. In some embodiments, the AAV2 rep gene contains the nucleotide sequence of SEQ ID NO: 63.

[0104] In some embodiments, the nucleotide sequences are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m 8. Encodes an AAV cap gene containing a serotype selected from the group consisting of AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the AAV cap gene contains a serotype selected from the group consisting of AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. In some embodiments, the AAV cap gene includes a serotype selected from the group consisting of AAV8 or AAV9 serotypes.

[0105] In some embodiments, the AAV cap gene contains the AAV2 serotype. In some embodiments, the AAV2 cap gene contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 64. In some embodiments, the AAV2 cap gene contains a nucleotide sequence having at least 90% identity with SEQ ID NO: 64. In some embodiments, the AAV2 cap gene contains a nucleotide sequence having at least 95% identity with SEQ ID NO: 64. In some embodiments, the AAV2 cap gene contains a nucleotide sequence having at least 98% identity with SEQ ID NO: 64. In some embodiments, the AAV2 cap gene contains the nucleotide sequence of SEQ ID NO: 64.

[0106] In some embodiments, the AAV cap gene contains the AAV6 serotype. In some embodiments, the AAV6 cap gene contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 65. In some embodiments, the AAV6 cap gene contains a nucleotide sequence having at least 90% identity with SEQ ID NO: 65. In some embodiments, the AAV6 cap gene contains a nucleotide sequence having at least 95% identity with SEQ ID NO: 65. In some embodiments, the AAV6 cap gene contains a nucleotide sequence having at least 98% identity with SEQ ID NO: 65. In some embodiments, the AAV6 cap gene contains the nucleotide sequence of SEQ ID NO: 65.

[0107] In some embodiments, the AAV cap gene contains the AAV8 serotype. In some embodiments, the AAV8 cap gene contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 66. In some embodiments, the AAV8 cap gene contains a nucleotide sequence having at least 90% identity with SEQ ID NO: 66. In some embodiments, the AAV8 cap gene contains a nucleotide sequence having at least 95% identity with SEQ ID NO: 66. In some embodiments, the AAV8 cap gene contains a nucleotide sequence having at least 98% identity with SEQ ID NO: 66. In some embodiments, the AAV8 cap gene contains the nucleotide sequence of SEQ ID NO: 66.

[0108] In some embodiments, the AAV cap gene contains the AAV9 serotype. In some embodiments, the AAV9 cap gene contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 67. In some embodiments, the AAV9 cap gene contains a nucleotide sequence having at least 90% identity with SEQ ID NO: 67. In some embodiments, the AAV9 cap gene contains a nucleotide sequence having at least 95% identity with SEQ ID NO: 67. In some embodiments, the AAV9 cap gene contains a nucleotide sequence having at least 98% identity with SEQ ID NO: 67. In some embodiments, the AAV9 cap gene contains the nucleotide sequence of SEQ ID NO: 67.

[0109] In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 68-70 or 71. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 90% identity with SEQ ID NOs. 68-70 or 71. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 95% identity with SEQ ID NOs. 68-70 or 71. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 98% identity with SEQ ID NOs. 68-70 or 71. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include the nucleotide sequences of SEQ ID NOs. 68-70 or 71.

[0110] Those skilled in the art will understand that the AAV nucleotide sequence of the AAV cap gene also encodes assembly-activating protein (AAP) and membrane-bound assembly protein (mAAP). Since the cap polypeptide is encoded in a different reading frame than the AAP and mAAP polypeptides, those skilled in the art will understand that the nucleotide sequence encoding the cap gene described herein may contain nonsense mutations that introduce stop codons into the reading frames encoding the AAP and mAAP polypeptides without disrupting the reading frames encoding the cap polypeptides. In some embodiments, the nucleotide sequence encoding the AAV cap gene includes mutations that affect the expression of the mAAP polypeptide. In some embodiments, the nucleotide sequence encoding the AAV cap gene includes mutations that introduce stop codons into the reading frame of the mAAP polypeptide, resulting in the production of cleaved mAAP polypeptides. In some embodiments, the nucleotide sequence encoding the AAV cap gene includes one or more mutations that introduce one or more stop codons into the reading frame of the mAAP polypeptide. In some embodiments, the nucleotide sequence encoding the AAV cap gene includes two, three, four, five, or six mutations, each introducing two, three, four, five, or six stop codons into the reading frame of the mAAP polypeptide. In some embodiments, the nucleotide sequence encoding the AAV cap gene contains six mutations that introduce six stop codons into the reading frame of the mAAP polypeptide. In some embodiments, one or more mutations affecting the mAAP reading frame include nonsense mutations to one or more of the amino acid residues S39, L78, E90, L100, L106, and L110 of mAAP. In some embodiments, one or more mutations affecting the mAAP reading frame include six nonsense mutations to the amino acid residues S39, L78, E90, L100, L106, and L110 of mAAP.In some embodiments, one or more mutations affecting the mAAP reading frame correspond to mutations present in any one of the pHRC#17-#34 packaging vectors. In some embodiments, mutations affecting the mAAP reading frame correspond to mutations present in the pHRC#38 or #39 packaging vector. In some embodiments, the nucleotide sequence encoding the AAV cap gene includes a mutation that disrupts the first start codon of the mAAP polypeptide. In some embodiments, the nucleotide sequence encoding the AAV cap gene includes a mutation that disrupts the translation initiation of the mAAP polypeptide. In some embodiments, the nucleotide sequence encoding the cap gene includes the nucleotide sequence encoding the cap gene of any one of the pHRC#17-#34 packaging vectors. In some embodiments, the nucleotide sequence encoding the cap gene includes the nucleotide sequence encoding the cap gene of the pHRC#38 or #39 packaging vector.

[0111] In some embodiments, the nucleotide sequence encoding the AAV cap gene contains one or more mutations that affect the expression of the mAAP polypeptide. In some embodiments, the AAV cap gene contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, or 261. In some embodiments, the AAV cap gene contains a nucleotide sequence having at least 90% identity with SEQ ID NOs: 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, or 261. In some embodiments, the AAV cap gene contains a nucleotide sequence having at least 95% identity with SEQ ID NOs: 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, or 261. In some embodiments, the AAV cap gene contains a nucleotide sequence having at least 98% identity to SEQ ID NOs. 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, or 261. In some embodiments, the AAV cap gene contains a nucleotide sequence of SEQ ID NOs. 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, or 261.

[0112] In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene contain one or more mutations that affect the expression of the mAAP polypeptide. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene contain nucleotide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 167, 172, 187, 192, 197, 202, 207, 212, 217, 222, 227, 232, 237, 242, 247, 252, 257, or 262. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 90% identity with SEQ ID NOs: 167, 172, 187, 192, 197, 202, 207, 212, 217, 222, 227, 232, 237, 242, 247, 252, 257, or 262. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 95% identity with SEQ ID NOs: 167, 172, 187, 192, 197, 202, 207, 212, 217, 222, 227, 232, 237, 242, 247, 252, 257, or 262. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences having at least 98% identity to SEQ ID NOs: 167, 172, 187, 192, 197, 202, 207, 212, 217, 222, 227, 232, 237, 242, 247, 252, 257, or 262. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include nucleotide sequences of SEQ ID NOs: 167, 172, 187, 192, 197, 202, 207, 212, 217, 222, 227, 232, 237, 242, 247, 252, 257, or 262.

[0113] Recombinant viral genome In some embodiments, the nucleotide sequence encoding the recombinant viral genome includes at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding the gene product, which is responsively bound to a sequence that directs the expression of the gene product in a target cell. In certain embodiments, the gene product is the gene product shown in Tables 2A-2C. In some embodiments, the rAAV genome includes the following components: (1) an AAV reverse terminal repeat adjacent to the expression cassette; (2) regulatory elements, e.g., a) a promoter / enhancer, b) a polyA signal, and c) an intron, optionally; and (3) a nucleic acid sequence encoding the transgene. In another embodiment for expressing intact or substantially intact monoclonal antibodies (mAbs), the rAAV genome comprises the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette; (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) optionally introns; and (3) nucleic acid sequences encoding the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally the heavy chain Fc region. In yet another embodiment for expressing intact or substantially intact mAbs, the rAAV genome comprises the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette;(2) Regulatory elements, e.g., a) promoters / enhancers, b) poly(A) signals, and c) introns (optionally), and (3) nucleic acid sequences encoding heavy chain Fab below: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., (Adalimumab, infliximumab, infliximumab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and salalizumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or their modified Fc;In addition, the following light chains: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidorumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab), anti-R GMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal The peptide includes mAbs (e.g., lanadermab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); and the heavy chain (e.g., Fab and optionally the Fc region) and light chain are separated by autocleaved furin (e.g., F) / F2A or a flexible linker, ensuring equal expression of heavy and light chain polypeptides.

[0114] In some embodiments, the isolated polynucleotides disclosed herein comprise a nucleotide sequence encoding a recombinant viral genome encoding anti-VEGF Fab, and optionally further comprising a nucleotide sequence encoding the AAV8 cap gene. In a more specific embodiment, anti-VEGF Fab is ranibizumab. In some embodiments, the isolated polynucleotides disclosed herein comprise a nucleotide sequence encoding a recombinant viral genome encoding idulonidase (IDUA), and optionally further comprising a nucleotide sequence encoding the AAV9 cap gene. In some embodiments, the isolated polynucleotides disclosed herein comprise a nucleotide sequence encoding a recombinant viral genome encoding idulonate 2-sulfatase (IDS), and optionally further comprising a nucleotide sequence encoding the AAV9 cap gene. In some embodiments, the isolated polynucleotides disclosed herein comprise a nucleotide sequence encoding a recombinant viral genome encoding a low-density lipoprotein receptor (LDLR), and optionally further comprising a nucleotide sequence encoding the AAV8 cap gene. In some embodiments, the isolated polynucleotides disclosed herein comprise a nucleotide sequence encoding a recombinant viral genome encoding the tripeptidyl peptidase 1 (TPP1) protein, and optionally, the isolated polynucleotides further comprise a nucleotide sequence encoding the AAV9 cap gene. In some embodiments, the isolated polynucleotides disclosed herein comprise a nucleotide sequence encoding a recombinant viral genome encoding a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, the isolated polynucleotides disclosed herein include gamma-sarcoglycans, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), and Tinocysin (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neuronurin (NRTN), neuronurin The recombinant viral genomes include nucleotide sequences encoding fin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-coded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusions.

[0115] Bocavirus NP1 and NS2 polypeptides In some embodiments, the isolated recombinant polynucleotides described herein further comprise nucleotide sequences encoding bocavirus NP1 and NS2 polypeptides. In some embodiments, the nucleotide sequences encoding bocavirus NP1 and NS2 polypeptides have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 12. In some embodiments, the nucleotide sequences encoding bocavirus NP1 and NS2 polypeptides have at least 90% identity with SEQ ID NO: 12. In some embodiments, the nucleotide sequences encoding bocavirus NP1 and NS2 polypeptides have at least 95% identity with SEQ ID NO: 12. In some embodiments, the nucleotide sequences encoding bocavirus NP1 and NS2 polypeptides have at least 98% identity with SEQ ID NO: 12. In some embodiments, the nucleotide sequences encoding bocavirus NP1 and NS2 polypeptides include SEQ ID NO: 12. In some embodiments, the bocavirus NP1 and NS2 polypeptides contain an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 52. In some embodiments, the bocavirus NP1 and NS2 polypeptides contain an amino acid sequence having at least 90% identity with SEQ ID NO: 52. In some embodiments, the bocavirus NP1 and NS2 polypeptides contain an amino acid sequence having at least 95% identity with SEQ ID NO: 52. In some embodiments, the bocavirus NP1 and NS2 polypeptides contain an amino acid sequence having at least 98% identity with SEQ ID NO: 52. In some embodiments, the bocavirus NP1 and NS2 polypeptides contain the amino acid sequence of SEQ ID NO: 52. In some embodiments, the nucleotide sequence encoding the bocavirus NP1 and NS2 polypeptides contains a CMV promoter.In some embodiments, the nucleotide sequences encoding the bocavirus NP1 and NS2 polypeptides include an engineered CMV early promoter. In some embodiments, the CMV early promoter includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 121. In some embodiments, the CMV early promoter includes the nucleotide sequence of SEQ ID NO: 121. Engineered CMV early promoters or their transcriptionally active fragments or portions are known to those skilled in the art, for example, as disclosed in International Application No. PCT / US2023 / 061014 (filed January 20, 2023) (the whole thereof is incorporated herein by reference).

[0116] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II and bocavirus NP1 and NS2 polypeptides. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 13. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 13. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 13. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 13.

[0117] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II and bocavirus NP1 and NS2 polypeptides. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 14. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 14. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 14. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 14. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 14.

[0118] In some embodiments, the isolated recombinant polynucleotides described herein include fragments comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6, nucleotide sequences encoding adenovirus VA RNA I and VA RNA II, and nucleotide sequences encoding bocavirus NP1 and NS2.

[0119] Adeno-associated virus assembly activating protein In some embodiments, the isolated recombinant polynucleotides described herein further comprise a nucleotide sequence encoding an adeno-associated virus (AAV) assembly-activating protein (AAP). Those skilled in the art will understand that the AAV AAP ORF overlaps with the AAV capsid ORF of the wild-type virus, and consequently, there exist AAV serotype-specific AAPs, e.g., AAP 1-13 corresponding to AAV serotypes 1-13. Sonntag et al., Journal of Virology, 85:12686-12697 (2011). In some embodiments, the AAP is AAP 1, AAP 2, AAP 3B, AAP 4, AAP 5, AAP 6, AAP 7, AAP 8, AAP 9, AAP 10, AAP 11, AAP 12, or AAV 13. In some embodiments, the AAP isotype matches the capsid isotype of the recombinant AAV produced. In some embodiments, the AAP is AAP 8. In some embodiments, AAP is AAP 9. In some embodiments, AAP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 53. In some embodiments, AAP comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the nucleotide sequence encoding AAV AAP has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding AAV AAP has at least 90% identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding AAV AAP has at least 95% identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding AAV AAP has at least 98% identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding AAV AAP includes SEQ ID NO: 15.In some embodiments, AAV AAP includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 53. In some embodiments, AAV AAP includes an amino acid sequence having at least 90% identity with SEQ ID NO: 53. In some embodiments, AAV AAP includes an amino acid sequence having at least 95% identity with SEQ ID NO: 53. In some embodiments, AAV AAP includes an amino acid sequence having at least 98% identity with SEQ ID NO: 53. In some embodiments, AAV AAP includes the amino acid sequence of SEQ ID NO: 53. In some embodiments, the nucleotide sequence encoding AAV AAP includes a CMV promoter. In some embodiments, the nucleotide sequence encoding AAV AAP includes an engineered CMV initial promoter. In some embodiments, the CMV initial promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 121. In some embodiments, the CMV initial promoter comprises the nucleotide sequence of SEQ ID NO: 121. Manipulated CMV initial promoters or their transcriptionally active fragments or portions are known to those skilled in the art, for example, as disclosed in International Application No. PCT / US2023 / 061014 (filed January 20, 2023) (the whole thereof is incorporated herein by reference).

[0120] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II and adeno-associated virus (AAV) assembly-activated protein (AAP). In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 16. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 16. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 16. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 16.

[0121] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II and adeno-associated virus (AAV) assembly-activated protein (AAP). In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 17. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 17. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 17. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 17.

[0122] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6, nucleotide sequences encoding adenovirus VA RNA I and VA RNA II, and a nucleotide sequence encoding adeno-associated virus (AAV) assembly-activating protein (AAP).

[0123] In some embodiments, the isolated recombinant polynucleotide described herein, comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 polypeptide, and nucleotide sequences encoding adenovirus VA RNA I and optionally VA RNA II, further comprises a nucleotide sequence encoding a membrane-bound assembly protein (mAAP). Those skilled in the art will understand that the mAAP ORF overlaps with the VP1 AAV capsid ORF in wild-type viruses, and consequently, AAV serotype-specific mAAPs exist. Overexpression of mAAPs can increase the yield of packaged viral particles. See, for example, International Publications WO2021 / 226253, WO2021260204, and WO2022046998 (each of which is incorporated herein by reference in whole). In some embodiments, the mAAP isotype matches the capsid isotype of the recombinant AAV produced.

[0124] Adenovirus E1A polypeptide In some embodiments, the isolated recombinant polynucleotides described herein further comprise a nucleotide sequence encoding the adenovirus E1A polypeptide. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide has at least 90% identity with SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide has at least 95% identity with SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide has at least 98% identity with SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide comprises SEQ ID NO: 18. In some embodiments, the adenovirus E1A polypeptide contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 51. In some embodiments, the adenovirus E1A polypeptide contains an amino acid sequence having at least 90% identity with SEQ ID NO: 51. In some embodiments, the adenovirus E1A polypeptide contains an amino acid sequence having at least 95% identity with SEQ ID NO: 51. In some embodiments, the adenovirus E1A polypeptide contains an amino acid sequence having at least 98% identity with SEQ ID NO: 51. In some embodiments, the E1A polypeptide contains the amino acid sequence of SEQ ID NO: 51. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide contains a CMV promoter. In some embodiments, the nucleotide sequence encoding the adenovirus E1A polypeptide contains an engineered CMV initial promoter.In some embodiments, the CMV initial promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 121. In some embodiments, the CMV initial promoter comprises the nucleotide sequence of SEQ ID NO: 121. Manipulated CMV initial promoters or their transcriptionally active fragments or portions are known to those skilled in the art, for example, as disclosed in International Application No. PCT / US2023 / 061014 (filed January 20, 2023) (the whole thereof is incorporated herein by reference).

[0125] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II and adenovirus E1A polypeptide. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity to SEQ ID NO: 19. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity to SEQ ID NO: 19. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity to SEQ ID NO: 19. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 19.

[0126] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, nucleotide sequences encoding adenovirus E4 ORF6 and ORF7 polypeptides, and nucleotide sequences encoding adenovirus VA RNA I and VA RNA II and adenovirus E1A polypeptide. In some embodiments, the fragment includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20. In some embodiments, the fragment includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 20. In some embodiments, the fragment includes a nucleotide sequence having at least 95% identity with SEQ ID NO: 20. In some embodiments, the fragment includes a nucleotide sequence having at least 98% identity with SEQ ID NO: 20. In some embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 20.

[0127] In some embodiments, the isolated recombinant polynucleotides described herein include a fragment comprising a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 ORF6, nucleotide sequences encoding adenovirus VA RNA I and VA RNA II, and a nucleotide sequence encoding adenovirus E1A polypeptide.

[0128] plasmid In some embodiments, this disclosure provides plasmids comprising the recombinant polynucleotide described herein, wherein the plasmid encodes one or more helper functions, an AAV rep gene, and an AAV cap gene, and the plasmid is capable of facilitating the generation of recombinant AAV particles within a host cell (e.g., HEK cells). In some embodiments, the plasmid described herein comprises a recombinant polynucleotide comprising (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding an adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, and (d) nucleotide sequences encoding an AAV rep gene and an AAV cap gene. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is immediately upstream of the nucleotide sequence encoding the AAV rep gene. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is separated from the nucleotide sequence encoding the AAV rep gene by about 1 to about 10,000 nucleotides. In some embodiments, the plasmid does not include nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursors.In some embodiments, the nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor are the corresponding nucleotide sequences of pAdDeltaF6. In some embodiments, the plasmid is a bacterial plasmid.

[0129] In some embodiments, the Disclosure provides plasmids comprising recombinant polynucleotides described herein, wherein the plasmid encodes one or more helper functions, an AAV rep gene, an AAV cap gene, and a recombinant AAV viral genome, and the plasmid is capable of facilitating the generation of recombinant AAV particles within a host cell (e.g., HEK cells). In some embodiments, the plasmids described herein include: (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP); (b) a nucleotide sequence encoding an adenovirus E4 polypeptide; (c) a nucleotide sequence encoding an adenovirus VA RNA I; (d) nucleotide sequences encoding an AAV rep gene and an AAV cap gene; and (e) a nucleotide sequence encoding a recombinant viral genome comprising at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product, which is responsively bound to a sequence directing the expression of the gene product in a target cell. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequence encoding the AAV rep gene and AAV cap gene comprises a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequence encoding the AAV rep gene and AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is immediately upstream of the nucleotide sequence encoding the AAV rep gene. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is separated from the nucleotide sequence encoding the AAV rep gene by about 1 to about 10,000 nucleotides.In some embodiments, the plasmid does not contain nucleotide sequences encoding an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor. In some embodiments, the nucleotide sequences encoding an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor are the corresponding nucleotide sequences of pAdDeltaF6. In some embodiments, the plasmid is a bacterial plasmid.

[0130] In some embodiments, the plasmids described herein include a bacterial origin of replication capable of growing the plasmid within a bacterial host cell (e.g., an E. coli host cell). In some embodiments, the bacterial origin of replication is a ColE1 origin.

[0131] In some embodiments, the plasmid described herein includes a selection marker gene. In some embodiments, the selection marker gene is a drug resistance gene. In some embodiments, the selection marker gene is a kanamycin resistance gene. In some embodiments, the selection marker gene is an ampicillin resistance gene.

[0132] In some embodiments, the plasmids described herein include a bacterial origin of replication and a selection marker gene.

[0133] In some embodiments, the plasmid described herein comprises (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding an adenovirus E4 polypeptide, (c) a nucleotide sequence encoding an adenovirus VA RNA I, (d) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene, (e) a nucleotide sequence encoding a parvovirus p5 promoter, and (f) a nucleotide sequence encoding an origin of replication and a selection marker gene, wherein the plasmid comprises the nucleotide sequence in the order 5'-(d)-(a)-(b)-(c)-(f)-3', and the parvovirus p5 promoter is actionably bound to the AAV rep gene and controls the expression of the rep78 and rep68 gene products. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is immediately upstream of the nucleotide sequence encoding the AAV rep gene. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is located about 1 to about 10,000 nucleotides upstream from the nucleotide sequence encoding the AAV rep gene. In some embodiments, the p5 promoter is located about 10 to about 10,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to about 4,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to about 2,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 2,000 to about 3,000 nucleotides upstream from the start codon of the AAV rep.In some embodiments, the p5 promoter is located approximately 3,000 to 4,000 nucleotides upstream from the start codon of the AAV rep gene. In some embodiments, the p5 promoter is located between nucleotide sequences (f) and (d). In some embodiments, the p5 promoter is located between nucleotide sequences (c) and (f). In some embodiments, the p5 promoter is located between nucleotide sequences (b) and (c). In some embodiments, the p5 promoter is located between the origin of replication and the selection marker gene. In some embodiments, the adenovirus E2A DBP coding region and the adenovirus E4 polypeptide coding region are in opposite 5'→3' directions. In some embodiments, the adenovirus E2A DBP coding region and the AAV rep gene and AAV cap gene coding regions are in opposite 5'→3' directions. In some embodiments, the adenovirus E2A DBP coding region and the adenovirus E4 polypeptide coding region are oriented in opposite 5'→3' directions, and the adenovirus E2A DBP coding region and the AAV rep gene and AAV cap gene coding regions are oriented in opposite 5'→3' directions. In some embodiments, the plasmid does not contain nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-associated precursor. In some embodiments, the plasmid contains about 10,000 to 15,000 nucleotides. In some embodiments, the plasmid contains about 10,000 to 14,000 nucleotides. In some embodiments, the plasmid contains about 10,000 to 13,000 nucleotides. In some embodiments, the plasmid contains about 12,000 to 13,000 nucleotides.

[0134] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with any one of the pHRC#1 to #39 packaging plasmids.

[0135] In some embodiments, the plasmids described herein include one nucleotide sequence from any of the pHRC#1 to #39 packaging plasmids.

[0136] In some embodiments, the plasmids described herein include a nucleotide sequence of a pHRC#5 or #7 packaging plasmid.

[0137] In some embodiments, the plasmids described herein include nucleotide sequences of pHRC#35, #36, or #37 packaging plasmids.

[0138] In some embodiments, the plasmids described herein include one nucleotide sequence from any of the pHRC#8-#16 packaging plasmids.

[0139] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs. 72-86 or 118.

[0140] In some embodiments, the plasmids described herein include the nucleotide sequences of SEQ ID NOs. 72-86 or 118.

[0141] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs. 72-86, 118, 159-161, 168, 173, 178, 183, 188, 193, 198, 203, 208, 213, 218, 223, 228, 233, 238, 243, 248, 253, 558, or 263.

[0142] In some embodiments, the plasmids described herein contain a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 76 or 77.

[0143] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs. 78-85 or 86.

[0144] In some embodiments, the plasmids described herein contain a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs. 159, 160, or 161.

[0145] In some embodiments, the plasmids described herein include the nucleotide sequences of SEQ ID NOs: 87-101, 119, 162-164, 169, 174, 179, 184, 189, 194, 199, 204, 209, 214, 219, 224, 229, 234, 239, 244, 249, 254, 259, or 264.

[0146] In some embodiments, the plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 91 or 92.

[0147] In some embodiments, the plasmids described herein include the nucleotide sequences of SEQ ID NOs. 93-100 or 101.

[0148] In some embodiments, the plasmids described herein include the nucleotide sequence of SEQ ID NO: 162, 163, or 164.

[0149] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs. 10, 11, 25-34, 56, 57, 106-108, or 109.

[0150] In some embodiments, the plasmids described herein include the nucleotide sequences of SEQ ID NOs: 10, 11, 25-34, 56, 57, 106-108, or 109.

[0151] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs. 110-116 or 117.

[0152] In some embodiments, the plasmids described herein include the nucleotide sequences of SEQ ID NOs. 110-116 or 117.

[0153] In some embodiments, the plasmid described herein comprises (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding an adenovirus E4 polypeptide, (c) a nucleotide sequence encoding an adenovirus VA RNA I, (d) a nucleotide sequence encoding the AAV rep gene and the AAV cap gene, (e) a nucleotide sequence encoding a parvovirus p5 promoter, (f) a nucleotide sequence encoding a recombinant AAV virus genome including a nucleic acid sequence encoding a gene product, and (g) a nucleotide sequence encoding the origin of replication and selection marker genes, wherein the plasmid comprises the nucleotide sequence in the order 5'-(d)-(a)-(b)-(f)-(c)-(g)-3', and the parvovirus p5 promoter is actionably bound to the AAV rep gene and controls the expression of the rep78 and rep68 gene products. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is located immediately upstream of the nucleotide sequence encoding the AAV rep gene. In some embodiments, the nucleotide sequence encoding the parvovirus p5 promoter is located about 1 to about 10,000 nucleotides upstream from the nucleotide sequence encoding the AAV rep gene. In some embodiments, the p5 promoter is located about 10 to about 10,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to about 4,000 nucleotides upstream from the start codon of the AAV rep. In some embodiments, the p5 promoter is located about 1,000 to about 2,000 nucleotides upstream from the start codon of the AAV rep.In some embodiments, the p5 promoter is located approximately 2,000 to 3,000 nucleotides upstream from the start codon of the AAV rep gene. In some embodiments, the p5 promoter is located approximately 3,000 to 4,000 nucleotides upstream from the start codon of the AAV rep gene. In some embodiments, the p5 promoter is located between nucleotide sequences (g) and (d). In some embodiments, the p5 promoter is located between nucleotide sequences (c) and (g). In some embodiments, the p5 promoter is located between nucleotide sequences (f) and (c). In some embodiments, the p5 promoter is located between the origin of replication and the selection marker gene. In some embodiments, the adenovirus E2A DBP coding region and the adenovirus E4 polypeptide coding region are in opposite 5'→3' directions. In some embodiments, the adenovirus E2A DBP coding region and the AAV rep gene and AAV cap gene coding regions are in opposite 5'→3' directions. In some embodiments, the adenovirus E2A DBP coding region and the adenovirus E4 polypeptide coding region are oriented in opposite 5'→3' directions, and the adenovirus E2A DBP coding region and the AAV rep gene and AAV cap gene coding regions are oriented in opposite 5'→3' directions. In some embodiments, the AAV rep gene and AAV cap gene coding regions and the gene product coding region contained by the recombinant AAV virus genome are oriented in opposite 5'→3' directions. In some embodiments, the plasmid does not contain nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-associated precursor. In some embodiments, the plasmid contains approximately 12,000 to 17,000 nucleotides. In some embodiments, the plasmid contains approximately 12,000 to 16,000 nucleotides. In some embodiments, the plasmid contains approximately 12,000 to 15,000 nucleotides. In some embodiments, the plasmid contains approximately 13,000 to 15,000 nucleotides.

[0154] In some embodiments, the plasmids described herein contain nucleotide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the complete plasmid of pHRCG#1, #2, #3, or #4, excluding the nucleotide sequences encoding the recombinant AAV virus genome, which include the nucleic acid sequence encoding the gene product. In some embodiments, the nucleotide sequences encoding the recombinant AAV virus genome within pHRCG#1, #2, #3, or #4 span the regions encoding the 5' and 3' ITRs and the sequences between the 5' and 3' ITRs.

[0155] In some embodiments, the plasmid comprises pHRCG#1, #2, #3, or #4 plasmids, excluding the nucleotide sequence encoding the recombinant AAV virus genome, which includes the nucleic acid sequence encoding the gene product. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome within pHRCG#1, #2, #3, or #4 spans the regions encoding the 5' and 3' ITRs and the sequence between the 5' and 3' ITRs.

[0156] In some embodiments, the plasmids described herein include a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs. 102, 103, 267, or 268, excluding the nucleotide sequence encoding the recombinant AAV virus genome, which includes the nucleic acid sequence encoding the gene product. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome spans the regions encoding the 5' and 3' ITRs and the sequence between the 5' and 3' ITRs.

[0157] In some embodiments, the plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 102, 103, 267, or 268, excluding the nucleotide sequence encoding the recombinant AAV virus genome, which includes the nucleic acid sequence encoding the gene product. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome spans the regions encoding the 5' and 3' ITRs and the sequence between the 5' and 3' ITRs.

[0158] In some embodiments, the plasmids described herein include a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs. 104, 105, 269, or 270, excluding the nucleotide sequence encoding the recombinant AAV virus genome, which includes the nucleic acid sequence encoding the gene product. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome spans the regions encoding the 5' and 3' ITRs and the sequences between the 5' and 3' ITRs. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome includes the nucleotide sequence of residues 9,537–14,270 of SEQ ID NOs. 104 or residues 9,526–14,259 of SEQ ID NOs. 105.

[0159] In some embodiments, the plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 104, 105, 269, or 270, excluding the nucleotide sequence encoding the recombinant AAV virus genome, which includes the nucleic acid sequence encoding the gene product. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome spans the regions encoding the 5' and 3' ITRs and the sequence between the 5' and 3' ITRs. In some embodiments, the nucleotide sequence encoding the recombinant AAV virus genome comprises the nucleotide sequence of residues 9,537–14,270 of SEQ ID NO: 104 or residues 9,526–14,259 of SEQ ID NO: 105.

[0160] In some embodiments, the plasmids described herein contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs. 110-116 or 117.

[0161] In some embodiments, the plasmids described herein include the nucleotide sequences of SEQ ID NOs. 110-116 or 117.

[0162] In some embodiments, the plasmids described herein contain nucleotide sequences having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the nucleotide sequences shown in Table 1.

[0163] [Table 1] TIFF2026513932000003.tif177165TIFF2026513932000004.tif135165

[0164] In some embodiments, the helper plasmids described herein include nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the E2A / E4 / VA helper functional sequences shown in Table 1. In some embodiments, the helper plasmids described herein include nucleotide sequences having the E2A / E4 / VA helper functional sequences shown in Table 1.

[0165] In some embodiments, the helper plasmids described herein include a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the helper plasmid "full plasmid" sequence shown in Table 1. In some embodiments, the helper plasmids described herein include a nucleotide sequence having the helper plasmid "full plasmid" sequence shown in Table 1.

[0166] In some embodiments, the packaging plasmid described herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the E2A / E4 / VA helper functional sequence shown in Table 1, a nucleotide sequence encoding the rep gene, and a nucleotide sequence encoding the cap gene. In some embodiments, the packaging plasmid further comprises a nucleotide sequence encoding the p5 promoter. In some embodiments, the packaging plasmid comprises a nucleotide sequence having the sequence of the E2A / E4 / VA helper functional sequence shown in Table 1. In some embodiments, the nucleotide sequence encoding the cap gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the cap gene sequence shown in Table 1. In some embodiments, the nucleotide sequence encoding the cap gene comprises the cap gene sequence shown in Table 1. In some embodiments, the nucleotide sequences encoding the rep gene and the nucleotide sequences encoding the cap gene include nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the rep / cap sequences shown in Table 1.

[0167] In some embodiments, the packaging plasmid described herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the pHRC "full plasmid" sequence shown in Table 1. In some embodiments, the packaging plasmid described herein comprises a nucleotide sequence having the pHCR "full plasmid" sequence shown in Table 1.

[0168] In some embodiments, the plasmids described herein are less than 15,000 bp in length. In some embodiments, the plasmids described herein are less than 13,000 bp in length. In some embodiments, the plasmids described herein are between 10,000 and 15,000 bp in length.

[0169] host cell In some embodiments, the disclosure provides host cells containing recombinant polynucleotides or plasmids as described herein. In some embodiments, the host cell is a prokaryotic cell capable of growing recombinant polynucleotides or plasmids as described herein. In some embodiments, the prokaryotic host cell is a bacterial cell. In some embodiments, the prokaryotic host cell is Escherichia coli. In some embodiments, the host cell is a eukaryotic cell capable of producing recombinant AAV particles. In some embodiments, the eukaryotic host cell is a mammalian cell. In some embodiments, the eukaryotic host cell is a HEK293 cell, an HEK-derived cell, a CHO cell, a CHO-derived cell, a HeLa cell, an SF-9 cell, a BHK cell, a Vero cell, or a PerC6 cell.

[0170] In some embodiments, the host cells described herein provide isolated recombinant polynucleotides encoding one or more helper functions and AAV rep and AAV cap genes, which can facilitate the generation of recombinant AAV particles within the host cell (e.g., HEK cell). In some embodiments, the host cell comprises isolated recombinant polynucleotides comprising (a) a nucleotide sequence encoding adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, and (d) nucleotide sequences encoding AAV rep and AAV cap genes. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the recombinant polynucleotide does not include nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor. In some embodiments, the plasmid is a bacterial plasmid.

[0171] In some embodiments, the host cell described herein comprises a recombinant polynucleotide encoding one or more helper functions, an AAV rep gene, and an AAV cap gene, and a recombinant AAV virus genome comprising a nucleic acid sequence encoding a gene product, the polynucleotide being capable of promoting the generation of recombinant AAV particles within the host cell (e.g., HEK cell). In some embodiments, the host cell comprises an isolated recombinant polynucleotide comprising (a) a nucleotide sequence encoding adenovirus E2A DBP, (b) a nucleotide sequence encoding adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, (d) nucleotide sequences encoding the AAV rep gene and the AAV cap gene, and (e) a nucleotide sequence encoding a recombinant AAV virus genome comprising a nucleic acid sequence encoding a gene product. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the nucleotide sequences encoding the AAV rep gene and the AAV cap gene include a nucleotide sequence encoding the parvovirus p5 promoter and the nucleotide sequences encoding the AAV rep gene and the AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 gene product and the rep68 gene product. In some embodiments, the recombinant polynucleotide does not include nucleotide sequences encoding the adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor. In some embodiments, the plasmid is a bacterial plasmid.

[0172] In some embodiments, the host cell described herein comprises a plasmid comprising (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding an adenovirus E4 polypeptide, (c) a nucleotide sequence encoding an adenovirus VA RNA I, (d) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene, (e) a nucleotide sequence encoding a parvovirus p5 promoter, and (f) a nucleotide sequence encoding an origin of replication and a selection marker gene, wherein the plasmid comprises a nucleotide sequence in the order 5'-(d)-(a)-(b)-(c)-(f)-3', and the parvovirus p5 promoter is actionably bound to the AAV rep gene and controls the expression of the rep78 and rep68 gene products. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the plasmid does not contain nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-associated precursors. In some embodiments, the plasmid is a bacterial plasmid.

[0173] In some embodiments, the host cell described herein comprises a plasmid comprising (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding an adenovirus E4 polypeptide, (c) a nucleotide sequence encoding an adenovirus VA RNA I, (d) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene, (e) a nucleotide sequence encoding a parvovirus p5 promoter, (f) a nucleotide sequence encoding a recombinant AAV virus genome including a nucleic acid sequence encoding a gene product, and (g) a nucleotide sequence encoding an origin of replication and a selection marker gene, wherein the plasmid comprises a nucleotide sequence in the order 5'-(d)-(a)-(b)-(f)-(c)-(g)-3', and the parvovirus p5 promoter is actionably bound to the AAV rep gene and controls the expression of the rep78 and rep68 gene products. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the plasmid does not contain nucleotide sequences encoding adenovirus ITR sequences, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-associated precursors. In some embodiments, the plasmid is a bacterial plasmid.

[0174] In some embodiments, the Disclosure provides a method for generating recombinant polynucleotides or plasmids as described herein, comprising incubating host cells as described herein under suitable conditions to generate recombinant polynucleotides or plasmids. In some embodiments, the host cells are prokaryotic cells capable of growing plasmids as described herein. In some embodiments, the prokaryotic host cells are bacterial cells. In some embodiments, the prokaryotic host cells are Escherichia coli.

[0175] Method for generating recombinant virus particles In one embodiment, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles in a eukaryotic host cell by using a recombinant polynucleotide, plasmid, or host cell described herein. In some embodiments, the method further includes recovering the rAAV particles.

[0176] In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising culturing cells capable of generating rAAV particles, wherein the cells comprise (i) a polynucleotide encoding an AAV capsid protein; (ii) a polynucleotide encoding a functional rep gene; (iii) a genome comprising a non-AAV nucleic acid sequence encoding a gene product, which is responsively bound to at least one AAV reverse terminal repeat (ITR) and a sequence directing the expression of the gene product in a target cell; and (iv) one or more polynucleotides comprising sufficient helper functions to enable packaging the genome into an AAV capsid protein under conditions that enable packaging the genome into an AAV capsid, wherein the recombinant polynucleotides or plasmids described herein comprise one or more helper functions, a recombinant polynucleotide encoding an AAV rep gene and an AAV cap gene, and optionally a recombinant AAV virus genome comprising a nucleic acid sequence encoding a gene product. In some embodiments, one or more helper functions include a nucleotide sequence encoding adenovirus E2A DBP, a nucleotide sequence encoding adenovirus E4 polypeptide, and a nucleotide sequence encoding adenovirus VA RNA I. In some embodiments, the adenovirus E4 polypeptide includes E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide includes E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the method further includes recovering rAAV particles.In some embodiments, the cell comprises (i) a polynucleotide disclosed herein, comprising (a) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP), (b) a nucleotide sequence encoding an adenovirus E4 polypeptide, (c) a nucleotide sequence encoding an adenovirus VA RNA I, and (d) nucleotide sequences encoding an AAV rep gene and an AAV cap gene, and (ii) a polynucleotide encoding a packaged rAAV genome. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the cell contains one polynucleotide disclosed herein, comprising (a) a nucleotide sequence encoding adenovirus E2A DBP, (b) a nucleotide sequence encoding adenovirus E4 polypeptide, (c) a nucleotide sequence encoding adenovirus VA RNA I, (d) a nucleotide sequence encoding the AAV rep gene and the AAV cap gene, and (e) a nucleotide sequence encoding a recombinant AAV virus genome including a nucleic acid sequence encoding the gene product. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the rAAV particle is an AAV8 or AAV9 particle. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have an AAV capsid protein with high sequence homology to AAV8 or AAV9, for example, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37.In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume of about 400 liters to about 5,000 liters.

[0177] In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing a cell culture containing cells; (b) introducing into cells a second polynucleotide comprising a genome containing a first nucleotide disclosed herein, comprising (i) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP); (ii) a nucleotide sequence encoding an adenovirus E4 polypeptide; (iii) a nucleotide sequence encoding an adenovirus VA RNA I; and (iv) nucleotide sequences encoding an AAV rep gene and an AAV cap gene, as well as a non-AAV nucleic acid sequence encoding a gene product, which is responsively bound to at least one AAV reverse terminal repeat (ITR) and a sequence that directs the expression of the gene product in a target cell; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing a cell culture comprising cells; (b) introducing a plasmid disclosed herein into the cells, comprising a polynucleotide comprising a genome including (i) a nucleotide sequence encoding an adenovirus E2A DNA-binding protein (DBP); (ii) a nucleotide sequence encoding an adenovirus E4 polypeptide; (iii) a nucleotide sequence encoding an adenovirus VA RNA I; (iv) nucleotide sequences encoding AAV rep and AAV cap genes, as well as a non-AAV nucleic acid sequence encoding a gene product, which is responsively bound to at least one AAV reverse terminal repeat (ITR) and a sequence that directs the expression of the gene product in a target cell; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7.In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I encodes adenovirus VA RNA I and VA RNA II. In some embodiments, the method further comprises recovering rAAV particles. In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume of about 400 liters to about 5,000 liters.

[0178] In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing a cell culture comprising cells; (b) introducing into the cells a polynucleotide disclosed herein, comprising: (i) a nucleotide sequence encoding adenovirus E2A DBP; (ii) a nucleotide sequence encoding adenovirus E4 polypeptide; (iii) a nucleotide sequence encoding adenovirus VA RNA I; (iv) a nucleotide sequence encoding AAV rep gene and AAV cap gene; and (v) a nucleotide sequence encoding a recombinant AAV virus genome comprising a nucleic acid sequence encoding a gene product; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing a cell culture containing cells; (b) introducing a plasmid disclosed herein into the cells, comprising: (i) a nucleotide sequence encoding adenovirus E2A DBP; (ii) a nucleotide sequence encoding adenovirus E4 polypeptide; (iii) a nucleotide sequence encoding adenovirus VA RNA I; (iv) a nucleotide sequence encoding the AAV rep gene and the AAV cap gene; and (v) a nucleotide sequence encoding a recombinant AAV virus genome, comprising a nucleic acid sequence encoding the gene product; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6. In some embodiments, the nucleotide sequence encoding adenovirus VA RNA I comprises adenovirus VA RNA I and VA RNA II. In some embodiments, the Method further comprises recovering the rAAV particles.In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have an AAV capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume of approximately 400 liters to approximately 5,000 liters.

[0179] In some embodiments, the methods disclosed herein involve introducing polynucleotides encoding AAV capsid proteins and functional rep genes into cells.

[0180] In some embodiments, the introduction of one or more polynucleotides and / or one or more plasmids into cells is carried out by transduction.

[0181] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, or PerC6 cells. In some embodiments, the cells are HEK293 cells.

[0182] In some embodiments, the cell culture is a suspension culture or an adherent culture.

[0183] In some embodiments, the cell culture has a volume between approximately 50 liters and approximately 20,000 liters.

[0184] In some embodiments, the method described herein produces more rAAV particles measured as GC / ml than the reference method. In some embodiments, the reference method uses a polynucleotide comprising a helper function comprising the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the reference method uses a polynucleotide comprising a helper function comprising the nucleotide sequence of SEQ ID NO: 44. In some embodiments, the method described herein produces at least about 10% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 10% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 20% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 30% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 40% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 50% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 70% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 90% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about twice as many rAAV particles measured as GC / ml as the reference method. In some embodiments, this method produces at least about three times more rAAV particles measured as GC / ml than the reference method. In some embodiments, this method produces at least about four times more rAAV particles measured as GC / ml than the reference method.

[0185] In some embodiments, this method generates a population of rAAV particles containing more complete capsids than the reference method. In some embodiments, the reference method uses a polynucleotide that includes a helper function containing the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the reference method uses a polynucleotide that includes a helper function containing the nucleotide sequence of SEQ ID NO: 44.

[0186] In some embodiments, rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m 8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype capsid proteins. In some embodiments, rAAV particles contain AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, or AAV.hu37 serotype capsid proteins. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0187] In some embodiments, the rAAV particle contains a transgene encoding a gene product. In some embodiments, the gene product is a polypeptide or a double-stranded RNA molecule. In some embodiments, the gene product is a polypeptide. In some embodiments, the transgene encodes an antibody or its antigen-binding fragment, a fusion protein, an Fc fusion polypeptide, an immunoadhesin, an immunoglobulin, a modified protein, a protein fragment, or an enzyme. In some embodiments, the transgene contains a regulatory element responsively bound to a polynucleotide encoding the gene product.

[0188] In some embodiments, the gene product is anti-VEGF Fab, anti-kallikrein antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, the gene products include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), and retinocytic sarcoglycan. (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurite The gene product is either dystrophin (NRTN), neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial code NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1)Fc fusion. In some embodiments, the gene product is dystrophin or microdystrophin.In some embodiments, the gene product is microRNA.

[0189] In some embodiments, the method described herein increases the generation of rAAV particles while maintaining or improving the quality attributes of rAAV particles and compositions containing them. In some embodiments, the quality of rAAV particles and compositions containing them is evaluated by quantifying the concentration of rAAV particles (e.g., GC / ml), the percentage of particles containing copies of the rAAV genome, the proportion of particles without the genome, the infectivity of the rAAV particles, the stability of the rAAV particles, and the concentration of residual host cell proteins or residual host cell nucleic acids (e.g., host cell genomic DNA, plasmids encoding rep and cap genes, plasmids encoding helper functions, plasmids encoding the rAAV genome). In some embodiments, the quality of rAAV particles or compositions containing them produced by the method described herein is the same as the quality of rAAV particles or compositions produced by a reference method using a helper plasmid containing the nucleotide sequence of SEQ ID NO: 35 or 44. In some embodiments, the quality of rAAV particles or compositions containing them produced by the method described herein is better than the quality of rAAV particles or compositions produced by a reference method using a helper plasmid containing the nucleotide sequence of SEQ ID NO: 35 or 44.

[0190] In the art, numerous cell culture-based systems for the generation of rAAV particles are known, and any of these can be used to carry out the methods described herein. An rAAV-generating culture for generating rAAV virus particles requires (1) suitable host cells (e.g., human cell lines (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), mammalian cell lines (e.g., Vero), CHO cells or CHO-derived cells); (2) suitable helper virus functions provided by wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide helper functions; (3) AAV rep and cap genes and gene products; (4) transgenes adjacent to the AAV ITR sequence (e.g., therapeutic transgenes); and (5) suitable media and media components to support rAAV generation.

[0191] Those skilled in the art are aware of numerous methods by which rAAV can be generated or packaged by introducing AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and rAAV genomes (containing one or more target genes adjacent to an inverse terminal repeat (ITR)) into cells. The expression "adenovirus helper function" means a set of viral helper genes that are expressed intracellularly (as RNA or protein) to enable AAV to grow efficiently within the cell. Those skilled in the art understand that helper viruses, including adenoviruses and herpes simplex virus (HSV), promote AAV replication, and that certain genes providing essential functions have been identified, and that, for example, helpers can induce changes in the cellular environment that promote such AAV gene expression and replication. In some embodiments of the methods described herein, the AAV rep and cap genes, helper genes, and rAAV genome are introduced into cells by transduction of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.

[0192] Molecular biology techniques for developing plasmids or viral vectors encoding AAV rep and cap genes, helper genes, and / or the rAAV genome are commonly known in the art. In some embodiments, the AAV rep and cap genes are encoded by a single plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single plasmid vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transduction with a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, helper genes are stably expressed by host cells. In some embodiments, the AAV rep and cap genes are encoded by a single viral vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single viral vector. In some embodiments, the E1a or E1b gene is stably expressed by host cells, and the remaining AAV helper genes are introduced into cells by transduction using a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by host cells, and the E4, E2a, and VA genes are introduced into cells by transduction using a single viral vector. In some embodiments, one or more helper genes are stably expressed by host cells, and one or more helper genes are introduced into cells by transduction using a single viral vector.In some embodiments, the AAV rep and cap genes, the adenovirus helper function required for packaging, and the rAAV genome to be packaged are introduced into cells by transduction using one or more polynucleotides, for example, a vector. In some embodiments, the method described herein involves transduction into cells of a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the AAV cap gene is the AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is the AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged contains the target gene adjacent to the AAV ITR.In some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, It is from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or another AAV serotype.

[0193] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and the rAAV genome into cells into which rAAV particles are generated or packaged. In some embodiments of the method described herein, a first plasmid vector encoding the rAAV genome containing a target gene adjacent to an AAV reverse terminal repeat (ITR), a second vector encoding the AAV rep and cap genes, and a third vector encoding the helper gene can be used. In some embodiments, a mixture of the three vectors can be simultaneously translocated into cells.

[0194] In some embodiments, a combination of translocation and infection is used by using a viral vector in conjunction with a plasmid vector.

[0195] In some embodiments, one or more of the rep and cap genes, as well as an AAV helper gene, are constitutively expressed by the cell and do not require transduction or transfection into the cell. In some embodiments, the cell constitutively expresses the rep and / or cap gene. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell contains a stable transgene encoding the rAAV genome.

[0196] In some embodiments, the AAV rep, cap, and helper genes (e.g., Ela gene, E1b gene, E4 gene, E2a gene, or VA gene) can be any AAV serotype. Similarly, the AAV ITR can also be any AAV serotype. For example, in some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, A AV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, A These are from AV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV cap gene is derived from the AAV9 or AAV8 cap gene.In some embodiments, the AAV cap gene is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF These are from AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV rep and cap genes for rAAV particle generation are derived from different serotypes. For example, the rep gene is derived from AAV2, while the cap gene is derived from AAV9.

[0197] Any suitable culture medium known in the art can be used to generate recombinant viral particles (e.g., rAAV particles) according to the method herein. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hydrone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety). In some embodiments, the medium includes Dynamis® medium, FreeStyle® 293 expression medium, or Expi293® expression medium from Invitrogen / ThermoFisher. In some embodiments, the medium includes Dynamis® medium. In some embodiments, the method herein uses cell cultures including serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the medium is animal component-free medium. In some embodiments, the medium includes serum. In some embodiments, the medium includes fetal bovine serum. In some embodiments, the culture medium is glutamine-free. In some embodiments, the culture medium contains glutamine. In some embodiments, the culture medium is supplemented with one or more of the following: nutrients, salts, buffers, and additives (e.g., antifoaming agents). In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium is supplemented with serum. In some embodiments, the culture medium is supplemented with fetal bovine serum. In some embodiments, the culture medium is supplemented with poloxamer, for example, Kolliphor® P 188 Bio. In some embodiments, the culture medium is a basic medium. In some embodiments, the culture medium is a feed medium.

[0198] Recombinant virus (e.g., rAAV) cultures can be routinely grown under a variety of conditions suitable for the specific host cell being used (over a wide temperature range, over varying lengths of time, etc.). As is known in the art, virus cultures can be adapted to suspension-adapted host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK- These include 1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, and SF-9 cells, which can be cultured in a variety of ways, including disposable systems such as spinner flasks, agitated tank bioreactors, and Wave bag systems. Numerous suspension cultures for generating rAAV particles are known in the art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole).

[0199] Any cell or cell line known in the art to produce recombinant virus particles (e.g., rAAV particles) can be used in any one of the methods described herein. In some embodiments, the methods for producing or increasing recombinant virus particles (e.g., rAAV particles) described herein use HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, LLC-MK cells, MDCK cells, RAF cells, RK cells, TCMK-1 cells, PK15 cells, BHK cells, BHK-21 cells, NS-1 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the methods described herein use mammalian cells. In some embodiments, the methods described herein use insect cells, such as SF-9 cells. In some embodiments, the methods described herein use cells adapted for growth in suspension culture. In some embodiments, the methods described herein use HEK293 cells adapted for growth in suspension culture.

[0200] In some embodiments, the cell cultures described herein are suspension cultures. In some embodiments, the large-scale suspension cell cultures described herein contain HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell cultures described herein contain serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell cultures described herein contain serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaking flask, spinner flask, cell bag, or bioreactor.

[0201] In some embodiments, the cell cultures described herein include serum-free media, animal component-free media, or chemically defined media. In some embodiments, the cell cultures described herein include serum-free media.

[0202] In some embodiments, the large-scale suspension cell cultures described herein include high-density cell cultures. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells.

[0203] The methods described herein can be used in the production of rAAV particles containing capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles contain capsid proteins from an AAV capsid serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles contain capsid proteins that are derivatives, modifications, or pseudotypes of the AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins.

[0204] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9.

[0205] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles comprise a capsid protein having a high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37.

[0206] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein having at least 80% identity, such as 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, i.e., having an AAV8 capsid protein.

[0207] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0208] In some embodiments, the rAAV particles contain capsid proteins having at least 80% identity to the VP1, VP2, and / or VP3 sequences of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, or AAV.7m8 capsid proteins, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity. In some embodiments, rAAV particles contain AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37, with at least 80% identity to the VP1, VP2, and / or VP3 sequences, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., capsid proteins with up to 100% identity.

[0209] In additional embodiments, the rAAV particles include a mosaic capsid. In additional embodiments, the rAAV particles include pseudotype rAAV particles. In additional embodiments, the rAAV particles include a capsid containing a capsid protein chimera of two or more AAV capsid serotypes.

[0210] rAAV particles The method provided is suitable for use in generating any isolated recombinant AAV particles. Therefore, rAAV can be any serotype, modifier, or derivative known in the art, or any combination thereof (e.g., a population of rAAV particles containing two or more serotypes (e.g., two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PH P.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more of these.

[0211] In some embodiments, rAAV particles are AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5 It possesses a capsid protein from an AAV serotype selected from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or their derivatives, modifications, or pseudotypes.In some embodiments, rAAV particles are, for example, AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.H The capsid protein contains a VP1, VP2, and / or VP3 sequence of an AAV capsid serotype selected from SC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 that is at least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0212] In some embodiments, rAAV particles are AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, This includes capsid proteins from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or their derivatives, modifiers, or a selection of AAV capsid serotypes from pseudotypes.In some embodiments, rAAV particles are, for example, AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HS It contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from C3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0213] In some embodiments, the rAAV particles comprise a capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068 (which is incorporated herein by reference in its entirety). In certain embodiments, the rAAV particles comprise a capsid having one of the amino acid inserts: LGETTRP or LALGETTRP, as described in U.S. Patents 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication 2016 / 0376323 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include the capsid of AAV.7m8, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323 (each of which is incorporated herein by reference in whole). In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,585,971, e.g., AAVPHP.B. In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313 (each of which is incorporated herein by reference in whole), e.g., AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2014 / 172669 (which is incorporated herein by reference in its entirety), e.g., AAV rh.74. In some embodiments, the rAAV particles include the AAV2 / 5 capsid as described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2017 / 070491 (which is incorporated herein by reference in its entirety), e.g., AAV2tYF.In some embodiments, the rAAV particles include a capsid of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418 (each of which is incorporated herein by reference in whole). In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 8,628,966, U.S. Patent No. 8,927,514, U.S. Patent No. 9,923,120, and WO2016 / 049230, e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of which is incorporated herein by reference in whole).

[0214] In some embodiments, rAAV particles are used in the following patents and patent applications (each of which is incorporated herein by reference in whole): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193, This includes AAV capsids disclosed in any of the following: Patent Nos. 956, 9458517, and 9,587,282; U.S. Patent Application Publications 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications (each of which is incorporated herein by reference in whole): for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical: U.S. Patents 7,282,199, 7,906,111, 8,524,446, 8,9 U.S. Patent Applications Nos. 99,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication No. U.S.2015 Patent applications No. / 0374803, No. 2015 / 0126588, No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, and international patent applications PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0215] In some embodiments, rAAV particles are used in International Patent Application Publications WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38), WO2009 / 1 The capsid protein is disclosed in Patent No. 04964 (see, for example, SEQ ID NOs. 1-5, 7, 9, 20, 22, 24, and 31), WO2010 / 127097 (see, for example, SEQ ID NOs. 5-38), and WO2015 / 191508 (see, for example, SEQ ID NOs. 80-294), and U.S. Patent Application Publication No. 20150023924 (see, for example, SEQ ID NOs. 1, 5-10) (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2 and / or VP3 sequences of the AAV capsid disclosed below, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., up to 100% identical: International Patent Application Publication WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 04239 See, for example, Sequence IDs 2, 81, 85, and 97; WO2006 / 068888 (see, for example, Sequence IDs 1 and 3-6); WO2006 / 110689 (see, for example, Sequence IDs 5-38); WO2009 / 104964 (see, for example, Sequence IDs 1-5, 7, 9, 20, 22, 24, and 31); WO2010 / 127097 (see, for example, Sequence IDs 5-38); and WO2015 / 191508 (see, for example, Sequence IDs 80-294); and U.S. Patent Application Publication No. 20150023924 (see, for example, Sequence IDs 1, 5-10).

[0216] Nucleic acid sequences of AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in: U.S. Patents Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patents Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publications 2015 / 0374803 and 2015 / 0126588. Patent applications No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, International Patent Application No. PCT / US2015 / 034799, No. PCT / EP2015 / 053335, No. WO2003 / 052051, No. WO2005 Patent applications Nos. / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, as well as U.S. Patent Application Publication No. 20150023924.

[0217] The provided method is suitable for use in generating recombinant AAV encoding a transgene. In certain embodiments, the transgenes are those shown in Tables 2A-2C. In some embodiments, the rAAV genome comprises a vector containing the following components: (1) an AAV reverse terminal repeat adjacent to the expression cassette, (2) regulatory elements, e.g., a) a promoter / enhancer, b) a polyA signal, and c) an intron (optionally), and (3) a nucleic acid sequence encoding the transgene. In other embodiments for expressing intact or substantially intact monoclonal antibodies (mAbs), the rAAV genome comprises a vector containing the following components: (1) an AAV reverse terminal repeat adjacent to the expression cassette, (2) regulatory elements, e.g., a) a promoter / enhancer, b) a polyA signal, and c) an intron (optionally), and (3) a nucleic acid sequence encoding the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally the heavy chain Fc region. In yet another embodiment for expressing intact or substantially intact mAbs, the rAAV genome comprises the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette;(2) Regulatory elements, e.g., a) promoters / enhancers, b) poly(A) signals, and c) introns (optionally), and (3) nucleic acid sequences encoding heavy chain Fab below: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., (Adalimumab, infliximumab, infliximumab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and salalizumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or their modified Fc;In addition, the following light chains: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidorumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab), anti-R GMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal The vector contains a mAb (e.g., lanadermab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); the heavy chain (e.g., Fab and optionally the Fc region) and light chain are separated by autocleaved furin (e.g., F) / F2A or a flexible linker, ensuring equal expression of heavy and light chain polypeptides.

[0218] [Table 2A] TIFF2026513932000006.tif176165TIFF2026513932000007.tif172165TIFF2026513932000008.tif175165TIFF2026513932000009.tif176165TIFF2026513932000010.tif181165TIFF2026513932000011.tif187165

[0219]

Table 2B

[0220]

Table 2C

[0221] In some embodiments, the rAAV particles are rAAV viral vectors encoding anti-VEGF Fab. In certain embodiments, the rAAV particles are rAAV8-based viral vectors encoding anti-VEGF Fab. In more specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding ranibizumab. In some embodiments, the rAAV particles are rAAV viral vectors encoding iduronidase (IDUA). In certain embodiments, the rAAV particles are rAAV9-based viral vectors encoding IDUA. In some embodiments, the rAAV particles are rAAV viral vectors encoding iduronate 2-sulfatase (IDS). In certain embodiments, the rAAV particles are rAAV9-based viral vectors encoding IDS. In some embodiments, the rAAV particles are rAAV viral vectors encoding low-density lipoprotein receptor (LDLR). In certain embodiments, the rAAV particles are rAAV8-based viral vectors encoding LDLR. In some embodiments, the rAAV particles are rAAV viral vectors encoding the tripeptidyl peptidase 1 (TPP1) protein. In certain embodiments, the rAAV particles are rAAV9-based viral vectors encoding TPP1. In some embodiments, the rAAV particles are rAAV viral vectors encoding a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, rAAV particles contain gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), and retinocysin (RS1). , sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (U GT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neuruturin (NRTN), nymph It is an rAAV virus vector encoding neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-coded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion.

[0222] In additional embodiments, the rAAV particles comprise a pseudotype AAV capsid. In some embodiments, the pseudotype AAV capsid is an rAAV2 / 8 or rAAV2 / 9 pseudotype AAV capsid. Methods for generating and using pseudotype rAAV particles are known in the art (see, for example, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0223] In additional embodiments, the rAAV particles comprise a capsid comprising a capsid protein that is a chimera of two or more AAV capsid serotypes. In some embodiments, the capsid protein is a chimera of two or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0224] In certain embodiments, single-stranded AAVs (ssAAVs) may be used. In certain embodiments, self-complementary vectors, such as scAAVs, may be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Patents 6,596,535, 7,125,717, and 7,456,683 (each of which is incorporated herein by reference in whole)).

[0225] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9.

[0226] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0227] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0228] In additional embodiments, the rAAV particles include a mosaic capsid. The mosaic AAV particles consist of a mixture of viral capsid proteins derived from different serotypes of AAV. In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, and AAV.PH It contains a mosaic capsid containing a capsid protein of a serotype selected from P.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles include a mosaic capsid containing a capsid protein of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0229] In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles include (a) a nucleic acid vector containing AAV ITR, and (b) AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, It contains a capsid composed of capsid proteins derived from AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). In additional embodiments, the rAAV particles include pseudotyped rAAV particles composed of capsid proteins of AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74. In additional embodiments, the rAAV particles include pseudotyped rAAV particles containing the AAV8 capsid protein. In additional embodiments, the rAAV particles include pseudotyped rAAV particles composed of the AAV9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles.Methods for generating and using pseudotyped rAAV particles are known in the art (see, for example, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001)).

[0230] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein that is a chimeric of two or more AAV capsid serotypes. In some embodiments, the rAAV particles contain the AAV8 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, It contains an AAV capsid protein that is a chimera with one or more AAV capsid proteins from AAV serotypes selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles contain an AAV capsid protein that is a chimera of the AAV8 capsid protein and one or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.In some embodiments, rAAV particles contain the AAV9 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B It contains an AAV capsid protein that is a chimera with a capsid protein of one or more AAV capsid serotypes selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles contain an AAV capsid protein that is a chimeric of the AAV9 capsid protein and the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0231] Method for isolating rAAV particles In some embodiments, the Disclosure provides a method for producing isolated recombinant adeno-associated virus (rAAV) particles, comprising isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture). In some embodiments, the method for producing a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles as described herein comprises (a) isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture), and (b) formulating the isolated rAAV particles to produce a formulation.

[0232] In some embodiments, the Disclosure further provides a method for producing a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles in a pharmaceutical unit dose, the method comprising isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture) and formulating the isolated rAAV particles.

[0233] The isolated rAAV particles can be isolated using methods known in the art. In some embodiments, the method for isolating rAAV particles includes downstream processing, e.g., collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, sterile filtration, or any combination(s) of these. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and sterile filtration. In some embodiments, the downstream process includes collecting the cell culture, clarifying the collected cell culture (e.g., by deep filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture by deep filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the clarification of the collected cell culture includes sterile filtration. In some embodiments, the downstream process does not include centrifugation. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0234] In some embodiments, a method for isolating rAAV particles according to the method described herein includes collecting a cell culture, clarifying the collected cell culture (e.g., by deep filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles produced according to the method herein includes clarification of the collected cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles produced according to the method described herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration.In some embodiments, the method for isolating rAAV particles described herein includes clarification of the collected cell culture by deep filtration, first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0235] In this field, numerous cell culture-based systems are known for translocation, stable cell line generation, and the production of rAAV particles, including infectious hybrid virus generation systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). Any rAAV-producing culture for generating rAAV virus particles requires (1) a suitable host cell (e.g., a human cell line (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), a mammalian cell line (e.g., Vero), or, in the case of a baculovirus-producing system, an insect cell line (e.g., SF-9)); (2) a suitable helper virus function provided by a wild-type or mutant adenovirus (e.g., a temperature-sensitive adenovirus), a herpesvirus, a baculovirus, or a plasmid construct providing helper function; (3) AAV rep and cap genes and gene products; (4) a transgene adjacent to the AAV ITR sequence (e.g., a therapeutic transgene); and (5) a suitable medium and medium components to support rAAV production. In some embodiments, the function of the suitable helper virus is provided by recombinant polynucleotides or plasmids as described herein. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety).

[0236] rAAV-producing cultures can be routinely grown under various conditions suitable for the specific host cells being used (over a wide temperature range, over varying lengths of time, etc.). As is known in the art, rAAV-producing cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers (e.g., roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors). Furthermore, rAAV vector-producing cultures can be grown with suspension-adaptive host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLC The cells may also include PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, agitated tank bioreactors, and disposable systems such as Wave bag systems. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted for growth in suspension culture. Numerous suspension cultures for generating rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole).

[0237] In some embodiments, the rAAV-producing culture comprises a high-density cell culture. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted for growth in suspension culture.

[0238] In additional embodiments of the provided method, the rAAV-producing culture includes a suspension culture containing rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole). In some embodiments, the suspension culture includes a culture of mammalian cells or insect cells. In some embodiments, the suspension culture includes cultures of HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the suspension culture includes a culture of HEK293 cells.

[0239] In some embodiments, a method for generating rAAV particles comprises preparing a cell culture containing cells capable of generating rAAV, adding a histone deacetylase (HDAC) inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM, and maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the HDAC inhibitor comprises a short-chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor comprises butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof.

[0240] In some embodiments, rAAV particles are generated as disclosed in WO2020 / 033842 (which is incorporated herein by reference in its entirety).

[0241] Recombinant AAV particles can be collected from rAAV-producing cultures by collecting the product culture containing host cells, or by collecting the consumed medium from the product culture, provided that the cells are cultured under conditions known in the art to induce the release of rAAV particles from intact host cells into the culture medium. Recombinant AAV particles can also be collected from rAAV-producing cultures by lysing the host cells of the product culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., surfactants and / or proteases).

[0242] At collection, rAAV-producing cultures may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper viruses; (5) helper virus proteins; (6) helper virus DNA; and (7) culture medium components (e.g., serum proteins, amino acids, transferrin, and other low molecular weight proteins). rAAV-producing cultures may also contain product-related impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

[0243] In some embodiments, the rAAV-producing culture collection is clarified to remove host cell debris. In some embodiments, the producing culture collection is clarified by filtration through a series of deep filters. Clarification can also be achieved by various other standard techniques known in the art, for example, by centrifugation or by filtration through any cellulose acetate filter with a pore size of 0.2 mm or larger known in the art. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the producing culture collection is clarified by centrifugation. In some embodiments, clarification of the producing culture collection does not include centrifugation.

[0244] In some embodiments, the collected cell culture is clarified using filtration. In some embodiments, the clarification of the collected cell culture includes deep filtration. In some embodiments, the clarification of the collected cell culture further includes deep filtration and sterile filtration. In some embodiments, the collected cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises one deep filtration medium. In some embodiments, the filter train comprises one or more deep filtration media. In some embodiments, the filter train comprises two deep filtration media. In some embodiments, the filter train comprises one sterile filtration medium. In some embodiments, the filter train comprises two deep filtration media and one sterile filtration medium. In some embodiments, the deep filtration medium is a porous deep filter. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® C0HC, and sterile-grade filter media. In some embodiments, the filter train includes Clarisolve® 20MS, Millistak+® C0HC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the collected cell culture is pretreated before contact with the deep filter. In some embodiments, the pretreatment includes adding salt to the collected cell culture. In some embodiments, the pretreatment includes adding a chemical flocculant to the collected cell culture. In some embodiments, the collected cell culture is not pretreated before contact with the deep filter.

[0245] In some embodiments, the collected culture is clarified by filtration, as disclosed in WO2019 / 212921 (which is incorporated herein by reference in its entirety).

[0246] In some embodiments, rAAV-producing culture collections are treated with a nuclease (e.g., Bensonase®) or endonuclease (e.g., endonuclease derived from Serratia marcescens) to digest the high molecular weight DNA present in the producing culture. Nuclease or endonuclease digestion can be routinely carried out under standard conditions known in the art. For example, nuclease digestion is carried out for 30 minutes to several hours with a final concentration of 1 to 2.5 units / mL of Bensonase® at temperatures ranging from ambient temperature to 37°C.

[0247] Aseptic filtration encompasses filtration using a sterile-grade filter medium. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile-grade filter medium contains polyethersulfone (PES). In some embodiments, the sterile-grade filter medium contains polyvinylidene fluoride (PVDF). In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 0.8 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 1.2 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In further embodiments, the sterile-grade filter medium is a 0.2 μm pore filter. In some embodiments, the sterile-grade filter medium is a combination of nominal pore sizes of Sartopore® 2 XLG 0.2 μm, Durapore® PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm + 0.2 μm. In some embodiments, the sterile-grade filter medium is Sartopore® 2 XLG 0.2 μm.

[0248] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatographic medium, such as an affinity chromatography medium. Large-scale concentrations of viruses using TFF ultrafiltration are described in Paul et al., Human Gene Therapy 4:609-615 (1993). The TFF concentration of the clarified feed allows for chromatographic application of technically controllable amounts of the clarified feed and enables more rational column sizing without requiring long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2x and at least 10x. In some embodiments, the clarified feed is concentrated between at least 10x and at least 20x. In some embodiments, the clarified feed is concentrated between at least 20x and at least 50x. In some embodiments, the clarified feed is concentrated to about 20x. Those skilled in the art will also recognize that TFF may be used to remove small molecule impurities (e.g., cell culture impurities including culture medium components, serum albumin, or other serum proteins) from a feed clarified via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 3 and about 10. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 5. Those skilled in the art will also recognize that TFF may be used at any step of the purification process when it is desirable to exchange the buffer before carrying out the next step in the purification process. In some embodiments, the method for isolating rAAV from a clarified feed described herein includes the use of TFF for buffer exchange.

[0249] Affinity chromatography can be used to isolate rAAV particles from a composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a feed that has been clarified by tangential flow filtration. Suitable affinity chromatography media include, but are not limited to, AVB Sepharose®, POROS® CaptureSelect® AAVX affinity resin, POROS® CaptureSelect® AAV9 affinity resin, and POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS® CaptureSelect® AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAVX affinity resin.

[0250] Anion exchange chromatography can be used to isolate rAAV particles from a composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polishing step. Suitable anion exchange chromatography media are known in the art and are not limited to, but include UNOsphere® Q (Biorad, Hercules, Calif.) and N-charged amino or imino resins, e.g., POROS® 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Patent No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media contains a quaternary amine. In some embodiments, the anion exchange medium is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises a glycidyl methacrylate-ethylenedimethacrylate polymer or a styrene-divinylbenzene polymer. In some embodiments, the monolithic anion exchange chromatography medium is selected from the group consisting of CIMmultus® QA-1 advanced composite column (quaternary amine), CIMmultus® DEAE-1 advanced composite column (diethylamino), CIM® QA disc (quaternary amine), CIM® DEAE, and CIM® EDA disc (ethylenediamino). In some embodiments, the monolithic anion exchange chromatography medium is a CIMmultus® QA-1 advanced composite column (quaternary amine). In some embodiments, the monolithic anion exchange chromatography medium is a CIM® QA disc (quaternary amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (column volume 80 mL). Those skilled in the art will understand that a wash buffer of suitable ionic strength can be identified so that impurities (including, but not limited to, impurities introduced by upstream purification steps) are removed while the rAAV maintains its binding to the resin.

[0251] In some embodiments, anion exchange chromatography is carried out according to the method disclosed in WO2019 / 241535 (which is incorporated herein by reference in its entirety).

[0252] In some embodiments, a method for isolating rAAV particles includes quantifying the vector genome titer, capsid titer, and / or the complete capsid:empty capsid ratio in a composition containing the isolated rAAV particles. In some embodiments, the vector genome titer is quantified by quantitative PCR (qPCR), digital PCR (dPCR), or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is quantified by serotype-specific ELISA. In some embodiments, the complete capsid:empty capsid ratio is quantified by analytical ultracentrifugation (AUC) or transmission electron microscopy (TEM).

[0253] In some embodiments, the vector genome titer, capsid titer, and / or the complete capsid:empty capsid ratio are quantified by spectrophotometric measurement, for example, by measuring the absorbance of the composition at 260 nm and at 280 nm. In some embodiments, the rAAV particles are not denatured before measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured before measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is quantified using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is quantified using HPLC. In some embodiments, the absorbance is peak absorbance. Several methods for measuring the absorbance of the composition at 260 nm and 280 nm are known in the art. A method for quantifying the vector genome titer and capsid titer of a composition containing isolated recombinant rAAV particles is disclosed in WO2019 / 212922 (which is incorporated herein by reference in its entirety).

[0254] In additional embodiments, the disclosure provides compositions comprising isolated rAAV particles produced according to the method described herein. In some embodiments, the compositions are pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

[0255] As used herein, the term “pharmaceutically acceptable” means a bioacceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, that can be administered to a subject without causing substantially undesirable biological effects. Such a pharmaceutical composition can therefore be used, for example, when administering rAAV isolated according to the methods of this disclosure to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil in water or water in oil), suspensions, syrups, elixirs, dispersions and suspension med...

Claims

1. Isolated recombinant polynucleotides, a) A first nucleotide sequence encoding adenovirus E2A DNA-binding protein (DBP), which is competently bound to the first promoter, b) A second nucleotide sequence encoding an adenovirus E4 polypeptide, which is competently bound to the second promoter, c) The third nucleotide sequence encoding adenovirus VA RNA I, d) A fourth nucleotide sequence encoding a parvovirus p5 promoter, which is optionally an adeno-associated virus (AAV) p5 promoter, and a fifth nucleotide sequence encoding an AAV rep gene and an AAV cap gene, wherein the parvovirus p5 promoter binds to the AAV rep gene and controls the expression of the rep78 and rep68 gene products. Includes, The isolated recombinant polynucleotide is optionally characterized in that the isolated recombinant polynucleotide does not contain nucleotide sequences encoding an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI / fiber, and / or L4 pVIII / hexone-related precursor.

2. a) The fourth nucleotide sequence encodes the AAV p5 promoter located 1,000 to 2,000 nucleotides upstream from the start codon of the AAV rep, and the isolated recombinant polynucleotide is a1) A fragment comprising the first nucleotide sequence encoding adenovirus E2A DBP and the second nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 282, a2) A fragment comprising the first nucleotide sequence encoding adenovirus E2A DBP, the second nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, and the third nucleotide sequence encoding adenovirus VA RNA I and VA RNA II, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 158, a3) A fragment comprising the first, second, third, and fifth nucleotide sequences, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 161, or a4) A nucleotide sequence having at least 95% identity with SEQ ID NO: 164 Includes, b) The fourth nucleotide sequence encodes the AAV p5 promoter located 1,000 to 2,000 nucleotides upstream from the start codon of the AAV rep, and the isolated recombinant polynucleotide is b1) A fragment comprising a first nucleotide sequence encoding adenovirus E2A DBP and a second nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 281, b2) A fragment comprising the first nucleotide sequence encoding adenovirus E2A DBP, the second nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, and the third nucleotide sequence encoding adenovirus VA RNA I and VA RNA II, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 157, b3) A fragment comprising the first, second, third, and fifth nucleotide sequences, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 160, or b4) A nucleotide sequence having at least 95% identity with SEQ ID NO: 163 including, or c) The fourth nucleotide sequence encodes the AAV p5 promoter located 2,000 to 3,000 nucleotides upstream from the start codon of the AAV rep, and the isolated recombinant polynucleotide is c1) A fragment comprising the first nucleotide sequence encoding adenovirus E2A DBP and the second nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 280, c2) A fragment comprising a first nucleotide sequence encoding adenovirus E2A DBP, a second nucleotide sequence encoding adenovirus E4 ORF6 polypeptide, and a third nucleotide sequence encoding adenovirus VA RNA I and VA RNA II, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 156, c3) A fragment comprising the first, second, third, and fifth nucleotide sequences, wherein the fragment comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 159, or c4) A nucleotide sequence having at least 95% identity with SEQ ID NO: 162 including, The isolated recombinant polynucleotide according to claim 1.

3. The isolated recombinant polynucleotide according to claim 1, wherein the nucleotide sequence encoding the adenovirus E2A DBP and the nucleotide sequence encoding the adenovirus E4 polypeptide are in opposite 5'→3' directions.

4. a) The adenovirus E2A DBP contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 45; b) The E4 polypeptide comprises the E4 ORF6 and ORF7, wherein the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 46, and the adenovirus E4 ORF7 polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 120; c) The E4 polypeptide comprises the E4 ORF6, and optionally the adenovirus E4 ORF6 polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 46; d) The nucleotide sequence encoding adenovirus VA RNA I includes a nucleotide sequence having at least 90% identity with SEQ ID NO: 54; and e) The nucleotide sequence encoding adenovirus VA RNA I includes, optionally, a nucleotide sequence encoding VA RNA I and VA RNA II, and having at least 90% identity with SEQ ID NO:

9. An isolated recombinant polynucleotide according to claim 1 or claim 3, characterized by one or more of the following.

5. a) The first promoter and the second promoter are different promoters; b) The first promoter is the adenovirus E2A promoter; and c) The second promoter is the adenovirus E4 promoter. An isolated recombinant polynucleotide according to claim 1, claim 3, or claim 4, characterized by one or more of the above.

6. a) The isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexone assembly polypeptide including an N-terminal deletion, and the E2A DBP, wherein the N-terminal deletion of the L4 100k / hexone assembly polypeptide corresponds to the nucleotide sequence of SEQ ID NO: 21, and optionally the nucleotide sequence has at least 90% identity with SEQ ID NO: 22; b) The isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100K / hexone assembly polypeptide having a mutation in its start codon, and the E2A DBP, and optionally the nucleotide sequence has at least 90% identity with SEQ ID NO: 23; c) The isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexone assembly polypeptide with an N-terminal deletion, and the E2A DBP, wherein the N-terminal deletion of the L4 100k / hexone assembly polypeptide includes the start codon of the L4 100k / hexone assembly but does not include the start codon of the L4 22K / 33K polypeptide; d) The isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexone assembly polypeptide including an N-terminal deletion, and the E2A DBP, wherein all or part of the L4 100k / hexone assembly polypeptide is deleted without disruption of the L4 22K / 33K start codon; e) The isolated recombinant polynucleotide comprises a nucleotide sequence encoding the E2A promoter, the L4 22K / 33K polypeptide and promoter, the L4 100k / hexone assembly polypeptide including an N-terminal deletion, and the E2A DBP, wherein the N-terminal deletion of the L4 100k / hexone assembly begins at the start codon of the L4 100k / hexone assembly and terminates immediately adjacent to the L4 22K / 33K promoter; f) The isolated recombinant polynucleotide contains a nucleotide sequence having at least 90% identity with SEQ ID NOs. 25-34, 56, 57, 106-109, 122-130, or 131; g) The isolated recombinant polynucleotide contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs: 140-158, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260; and h) The isolated recombinant polynucleotide contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 265 or 266. An isolated recombinant polynucleotide according to any one of claims 1 and 3 to 5, characterized by one or more of the above.

7. a) The AAV rep gene and the AAV cap gene have the same serotype; b) The AAV rep gene and the AAV cap gene have different serotypes; c) The AAV rep gene includes the AAV2 rep gene; d) The AAV cap gene includes a serotype selected from the group consisting of AAV8, AAV9, AAV. rh10, AAV. rh20, AAV. rh39, AAV. rh74, AAV. RHM4-1, AAV. hu32, and AAV. hu37; e) The AAV cap gene includes a serotype selected from the group consisting of AAV8 or AAV9 serotypes; f) The AAV cap gene includes the AAV2, AAV6, AAV8, or AAV9 serotype; g) The sequence encoding the cap gene contains one or more mutations that disrupt the expression of the mAAP polypeptide; h) The sequence encoding the cap gene contains one or more nonsense mutations within the mAAP ORF that disrupt the expression of the mAAP polypeptide; and / or i) The sequence encoding the cap gene includes a mutation in the start codon of the mAAP ORF that disrupts the expression of the mAAP polypeptide, An isolated recombinant polynucleotide according to any one of claims 1 and 3 to 6.

8. a) The p5 promoter is located approximately 10 to 10,000 nucleotides upstream from the start codon of the AAV rep; b) The p5 promoter is located approximately 5,000 to 10,000 nucleotides upstream from the start codon of the AAV rep; c) The p5 promoter is located approximately 1,000 to 5,000 nucleotides upstream from the start codon of the AAV rep; d) The first, second, and / or third nucleotide sequences are located between the p5 promoter upstream of the start codon of the AAV rep and the start codon of the AAV rep; e) The first, second, and third nucleotide sequences are located between the p5 promoter upstream of the start codon of the AAV rep and the start codon of the AAV rep; and / or f) The first, second, or third nucleotide sequence is not located between the p5 promoter upstream of the start codon of the AAV rep and the start codon of the AAV rep. An isolated recombinant polynucleotide according to any one of claims 1 and 3 to 7.

9. a) Nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence numbers 72-86, 118, 159-161, 168, 173, 178, 183, 188, 193, 198, 203, 208, 213, 218, 223, 228, 233, 238, 243, 248, 253, 558, or 263, b) A nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 76 or 77, c) Nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence numbers 87-101, 119, 162-164, 169, 174, 179, 184, 189, 194, 199, 204, 209, 214, 219, 224, 229, 234, 239, 244, 249, 254, 259, or 264, or d) Nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 91 or 92. An isolated recombinant polynucleotide according to any one of claims 1 and 3 to 8, comprising

10. An isolated recombinant polynucleotide according to any one of claims 1 and 3 to 9, which is a plasmid containing a bacterial origin of replication and a selection marker gene.

11. a) The bacterial replication origin or the selection marker gene is located between the fourth nucleotide and the fifth nucleotide such that the transcription initiated by the p5 promoter crosses the bacterial replication origin or the selection marker gene before reaching the AAV rep gene, or b) The bacterial origin of replication and the selection marker gene are located between the fourth nucleotide and the fifth nucleotide such that the transcription initiated by the p5 promoter crosses the bacterial origin of replication and the selection marker gene, respectively, before reaching the AAV rep gene. The isolated recombinant polynucleotide according to claim 10.

12. A sixth nucleotide sequence encoding a recombinant viral genome, comprising at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding the gene product, which is responsively bound to a sequence that directs the expression of the gene product in a target cell. An isolated recombinant polynucleotide according to any one of claims 1 and 3 to 11, comprising

13. The cells can be optionally selected from HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, or PerC6 cells. A host cell comprising an isolated recombinant polynucleotide according to any one of claims 1 to 12.

14. Incubate the host cell described in claim 13 under suitable conditions. A method for producing isolated recombinant polynucleotides according to any one of claims 1 to 12, comprising:

15. A method for generating rAAV particles, a) Prepare a cell culture containing cells, b) The cells, i. The polynucleotide according to any one of claims 1 to 11, and ii. A polynucleotide comprising a genome containing at least one AAV reverse terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding the gene product, which is competently bound to a sequence that directs the expression of the gene product in a target cell. Introducing and c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. Includes, The introduction of the polynucleotide into the cell is performed by transfusion, at the discretion of the user. The cells may be optionally HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, or PerC6 cells. Optionally, the gene product may be a polypeptide or a double-stranded RNA molecule. Optionally, the gene product is dystrophin or microdystrophin. The aforementioned method.

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