WO2025090962A1

WO2025090962A1 - Compositions and methods for recombinant aav production

Info

Publication number: WO2025090962A1
Application number: PCT/US2024/053113
Authority: WO
Inventors: Metewo Selase ENUAMEH; Ping Liu; Chih-Li Lin; Yusuf JOHARI; Thilo H. POHLE; David C. James
Original assignee: Regenxbio Inc
Current assignee: Regenxbio Inc
Priority date: 2023-10-25
Filing date: 2024-10-25
Publication date: 2025-05-01
Anticipated expiration: 2026-04-25

Abstract

Provided herein are recombinant polynucleotides, polynucleotide vectors and recombinant cells suitable for use in the production of recombinant AAV particles. Also provided herein are methods for producing rAAV particles.

Description

COMPOSITIONS AND METHODS FOR RECOMBINANT AAV PRODUCTION

TECHNICAL FIELD

[0001] The present disclosure relates to recombinant polynucleotides, recombinant cells comprising a stably integrated polynucleotide, and methods of producing recombinant adeno- associated virus (rAAV) particles.

CROSS-REFRENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. application no. 63/593,220, filed October 25, 2023, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The content of the electronically submitted sequence listing (Name: 6728_2301_Sequence_Listing.xml; Size: 442,445 bytes; and Date of Creation: October 24, 2024) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND

[0004] Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used gene therapy products in development. The preferred use of rAAV vector systems is due, in part, to the lack of disease associated with the wild-type virus, the ability of AAV to transduce non-dividing as well as dividing cells, and the resulting long-term robust transgene expression observed in clinical trials and that indicate great potential for delivery in gene therapy indications. Additionally, different naturally occurring and recombinant rAAV vector serotypes, specifically target different tissues, organs, and cells, and help evade any pre-existing immunity to the vector, thus expanding the therapeutic applications of AAV-based gene therapies. Before gene therapies based on a replication defective virus, for example, AAV can be more widely adopted for late clinical stage and commercial use. new methods for large scale production of recombinant virus particles need to be developed.

[0005] The triple plasmid transfection system in HEK293 cells is well established and commonly used for clinical and commercial manufacturing. In this system, one plasmid, often referred to as the trans plasmid, carries Rep and Cap genes and encodes proteins for virus replication and capsid formation. A second plasmid, often referred to as the helper plasmid, encodes the essential adenovirus helper genes (E4, E2A, and viral associated (VA) RNAs), and a third plasmid, often referred to as the cis plasmid, contains an expression cassette flanked by two inverted terminal repeats (ITRs), which is incorporated into the rAAV as its genome. The additional helper genes E1A and E1B are expressed endogenously by the HEK293 cells. The El A protein increases Rep protein expression by transactivating the AAV P5 and P19 promoters, and tire Rep protein initiates AAV replication.

[0006] Stable recombinant producer cell lines that express polypeptides necessary for recombinant AAV production could further reduce the cost of rAAV production and promote the wider adoption of rAAV for late clinical stage and commercial use. A consistent problem facing the development of stable rAAV production cell lines is that several polypeptides necessary for rAAV production are toxic to the cells (Qiao, C. et al. Gene Therapy 2002, Volume 76, Issue 24, https://doi.org/10. 1128/jvi.76.24. 13015-13027.2002). Another difficulty for stable rAAV producer cell is achieving the coordinated stable expression of tire at least 10 polypeptides necessary for efficient rAAV production (Martin, J. et al. Human Gene Therapy Methods 2013 24:4, 253-269). Thus, there is a need in the art to improve the productivity and yield of methods for the large-scale production of rAAV particles by providing improved polynucleotides encoding rAAV helper and packaging functions and recombinant rAAV producing cells comprising the polynucleotides stably integrated into their genome.

BRIEF SUMMARY

[0007] In one aspect, the disclosure provides an isolated recombinant polynucleotide encoding one or more helper functions, one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the first and second polynucleotides are comprised by a packaging polynucleotide. In some embodiments, an isolated recombinant polynucleotide described herein encodes one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, an isolated recombinant polynucleotide described herein encodes one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. [0008] In one aspect, the disclosure provides a polynucleotide vector comprising a recombinant polynucleotide described herein. In some embodiments, the recombinant polynucleotide described herein encodes one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP). a gene expressing an adenovirus E4 polypeptide (e.g.. an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the isolated recombinant polynucleotide described herein encodes one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the one or more AAV rep genes and/or AAV cap gene is an inducible gene.

[0009] In one aspect, the disclosure provides a recombinant cell comprising a recombinant polynucleotide or polynucleotide vector described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell comprises a recombinant polynucleotide or polynucleotide vector described herein encoding one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the cell comprises a recombinant polynucleotide or polynucleotide vector described herein encoding one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the cell comprises a recombinant polynucleotide or polynucleotide vector described herein encoding one or more helper functions and a recombinant polynucleotide or polynucleotide vector described herein encoding one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the cell comprises a stably integrated recombinant polynucleotide or polynucleotide vector described herein encoding one or more helper functions and/or a stably integrated recombinant polynucleotide or polynucleotide vector described herein encoding one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, a recombinant cell described herein further comprises a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell. In some embodiments, a cell disclosed herein is capable of producing an rAAV particle.

[0010] In one aspect, the disclosure provides a method of producing rAAV particles using the recombinant polynucleotides, polynucleotide vectors and/or recombinant cells described herein.

[0011] In one aspect, the disclosure provides a method of producing the recombinant polynucleotides, polynucleotide vectors and recombinant cells described herein.

[0012] In some embodiments, the disclosure provides:

[Embodiment 1.] A recombinant mammalian cell comprising a first and/or second stably integrated polynucleotide wherein a) the first polynucleotide encodes an adenovirus E2A DNA binding protein (DBP). an adenovirus E4 polypeptide and an adenovirus VA RNA gene; and b) the second polynucleotide comprises an AAV rep gene and an AAV cap gene, optionally wherein the first and second polynucleotides arc comprised by a packaging polynucleotide.

[Embodiment 2.] Tire recombinant cell of embodiment 1. wherein the first polynucleotide comprises, in a 5' to 3' order, a) a first promoter operably linked to a polynucleotide encoding the adenovirus E4 polypeptide; b) a second promoter operably linked to a polynucleotide encoding the adenovirus E2A DNA binding protein (DBP); and c) a polynucleotide encoding the adenovirus VA RNA gene; wherein the E4 polypeptide comprises the E4 ORF1-6/7, E4 ORF2-6/7, E4 ORF3-6/7, E4 ORF4- 6/7, E4 ORF6-6/7, E4 ORF6, or E4 ORF3+6-6/7.

[Embodiment 3.] The recombinant cell of embodiment 2, wherein the E4 polypeptide comprises the E4 ORF6-6/7, E4 ORF6, or E4 ORF3+6-6/7.

[Embodiment 4.] Hie recombinant cell of embodiment 2. wherein the E4 polypeptide comprises the E4 ORF6 or E4 ORF6/7.

[Embodiment 5.] The recombinant cell of embodiment 2 to embodiment 4, wherein the E4 ORF1, ORF2, ORF3, ORF4, ORF6 and ORF6/7 polypeptide comprises an amino acid sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10-14 and 15, respectively.

[Embodiment 6.] The recombinant cell of embodiment 2 to embodiment 4, wherein the polynucleotide encoding the adenovirus E4 poly peptide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16-21 or 22.

[Embodiment 7.] The recombinant cell of embodiment 6. wherein the polynucleotide encodes the adenovirus E4 OR6 or ORF6-6/7 polypeptide and comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16 or 21 . [Embodiment 8.] The recombinant cell of any one of embodiment 2 to embodiment 7, wherein the first promoter is an inducible promoter.

[Embodiment 9.] Tire recombinant cell of embodiment 8. wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 10 ] The recombinant cell of embodiment 9, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

[Embodiment 11.] Hie recombinant cell of any one of embodiment 2 to embodiment 10, wherein the E2A DBP comprises an amino acid sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8.

[Embodiment 12 ] The recombinant cell of any one of embodiment 2 to embodiment 10, wherein the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6 or 7.

[Embodiment 13.] Hie recombinant cell of any one of embodiment 2 to embodiment 12, wherein the second promoter is an inducible promoter.

[Embodiment 14.] The recombinant cell of embodiment 13, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 15.] The recombinant cell of embodiment 14, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1. 2 or 3.

[Embodiment 16.] Hie recombinant cell of any one of embodiment 2 to embodiment 15, wherein the first and second promoters are different. [Embodiment 17 ] The recombinant cell of any one of embodiment 2 to embodiment 15, wherein the first and second promoters are the same, optionally wherein the first and second promoters comprise a bidirectional promoter.

[Embodiment 18.] Hie recombinant cell of any one of embodiment 2 to embodiment 17, wherein the first polynucleotide further comprises a promoter operably linked to an adenovirus L4 22K/33K polypeptide coding region.

[Embodiment 19 ] The recombinant cell of embodiment 18, wherein the promoter operably linked to the adenovirus L4 22K/33K polypeptide coding region is an adenovirus L4 22K733K promoter or an inducible promoter, optionally a tetracycline inducible promoter.

[Embodiment 20.] Hie recombinant cell of any one of embodiment 2 to embodiment 19, wherein the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least about 90%. 95%. 97%. 98%. 99% or 100% identity to SEQ ID NO: 30. [Embodiment 21 .] The recombinant cell of any one of embodiment 2 to embodiment 20, wherein the first polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 33-38 or 39.

[Embodiment 22.] Hie recombinant cell of any one of embodiment 1 to embodiment 21, wherein the second polynucleotide comprises a third promoter operably linked to a third polynucleotide comprising, in 5' to 3' order an AAV cap coding region, an internal ribosome entry site (IRES), and a first AAV rep coding region, wherein a) the AAV cap coding region encodes a VP1, VP2 and VP3 polypeptide; b) the first AAV rep coding region encodes one or more of a Rcp78, Rcp68, Rcp52 and Rep40 polypeptide; c) the first AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity.

[Embodiment 23 ] The recombinant cell of embodiment 22, wherein the first AAV rep coding region encodes a (i) a Rep 78 and Rep 52 polypeptide, (ii) Rep 68 and Rep 40 polypeptide, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptide, wherein the first AAV rep coding region comprises a functional pl9 promoter.

[Embodiment 24.] Hie recombinant cell of embodiment 22, wherein the first AAV rep coding region encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide and wherein the AAV rep coding region comprises a functional pl9 promoter. [Embodiment 25 ] The recombinant cell of embodiment 22, wherein the first AAV rep coding region encodes a (i) a Rep78 polypeptide, (ii) Rep 68, or (iii) a Rep78 and Rep 68 polypeptide and wherein the first AAV rep coding region comprises a pl9 promoter comprising a mutation that reduces or eliminates its activity.

[Embodiment 26.] Tire recombinant cell of embodiment 22, wherein the first AAV rep coding region encodes a Rep78 and Rep 68 polypeptide.

[Embodiment 27 ] The recombinant cell of any one of embodiment 22 to embodiment 26, further comprising a seventh polynucleotide comprising a seventh promoter and a second Rep coding region, wherein the second Rep coding region encodes (i) a Rep 52 polypeptide, (ii) Rep40 polypeptide or (iii) a Rep52 and Rep40 polypeptide, wherein the second rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity.

[Embodiment 28. J The recombinant cell of embodiment 27, wherein the second Rep coding region encodes a Rep52 and Rep40 polypeptide.

[Embodiment 29.] The recombinant cell of embodiment 27 or embodiment 28, wherein seventh promoter is an inducible promoter.

[Embodiment 30.] Hie recombinant cell of embodiment 29, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 31.] The recombinant cell of embodiment 35, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

[Embodiment 32.] The recombinant cell of any one of embodiment 22 to embodiment 31, wherein the third promoter is an inducible promoter.

[Embodiment 33.] The recombinant cell of embodiment 32, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 34 ] The recombinant cell of embodiment 33, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

[Embodiment 35.] Hie recombinant cell of any one of embodiment 22 to embodiment 34, wherein the IRES comprises a nucleotide sequence having at least about 90%, 95%. 97%. 98%. 99% or 100% identity to SEQ ID NO: 69. [Embodiment 36 ] The recombinant cell of any one of embodiment 22 to embodiment 45, wherein the third polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 70 or 72.

[Embodiment 37.] Hie recombinant cell of any one of embodiment 22 to embodiment 36, wherein the second polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 71 or 73.

[Embodiment 38 ] The recombinant cell of any one of embodiment 1 to embodiment 21 , wherein the second polynucleotide comprises a) a fourth promoter operably linked to a fourth polynucleotide comprising in 5' to 3' order i. a polynucleotide encoding the 5' portion of an AAV Rep78 and/or 68 polypeptide, ii. an intron comprising a fifth promoter, and iii. a fifth polynucleotide comprising overlapping coding regions encoding the 3' portion of the AAV Rep78 and/or 68 polypeptide and an AAV Rep52 and/or Rep40 polypeptide, wherein

- the fifth promoter is operably linked to the fifth polynucleotide;

- the fourth polynucleotide encodes (i) a Rep78 and Rep52 polypeptides; (ii) a Rep68 and Rep40 polypeptides, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptides,

- and each of the p 19 and p40 promoters comprises a mutation that reduces or eliminates their activity; and b) a sixth promoter operably linked to a polynucleotide comprising a AAV cap coding region, wherein the AAV cap coding region encodes a VP1. VP2 and VP3 polypeptide. [Embodiment 39.] Tire recombinant cell of embodiment 38, wherein the fourth polynucleotide encodes a Rep78 and Rep52 polypeptides.

[Embodiment 40 ] The recombinant cell of embodiment 38 or embodiment 39, wherein the fourth promoter is an inducible promoter.

[Embodiment 41.] The recombinant cell of embodiment 40, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 42.] Tire recombinant cell of embodiment 46, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 2. [Embodiment 43 ] The recombinant cell of any one of embodiment 38 to embodiment 42, wherein the fifth promoter is an inducible promoter.

[Embodiment 44.] The recombinant cell of embodiment 43, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 45.] Tire recombinant cell of embodiment 49, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%. 95%. 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 2.

[Embodiment 46.] The recombinant cell of any one of embodiment 38 to embodiment 45, wherein the fifth and sixth promoters are different.

[Embodiment 47.] Hie recombinant cell of any one of embodiment 38 to embodiment 45, wherein the fifth and sixth promoters are the same.

[Embodiment 48. J The recombinant cell of any one of embodiment 38 to embodiment 47. wherein the intron comprising the fifth promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 57, 58, 59 or 60.

[Embodiment 49.] The recombinant cell of any one of embodiment 38 to embodiment 48, wherein the fourth polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 61, 62, 63 or 64.

[Embodiment 50.] The recombinant cell of any one of embodiment 38 to embodiment 49, wherein the sixth promoter is an inducible promoter.

[Embodiment 51.] The recombinant cell of embodiment 50, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 52.] Hie recombinant cell of embodiment 50, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 2.

[Embodiment 53 ] The recombinant cell of any one of embodiment 38 to embodiment 49, wherein the sixth promoter is constitutive promoter.

[Embodiment 54.] The recombinant cell of embodiment 53, wherein the sixth promoter is a CMV promoter.

[Embodiment 55.] Hie recombinant cell of embodiment 54, wherein the sixth promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4 or 76. [Embodiment 56 ] The recombinant cell of any one of embodiment 38 to embodiment 55, wherein the second polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82.

[Embodiment 57.] Hie recombinant cell of any one of embodiment 1 to embodiment 21, wherein the second polynucleotide comprises in 5' to 3' order an eighth promoter operably linked to a first AAV Rep coding region and a ninth promoter operably linked to an AAV Cap coding region, wherein a) the first AAV rep coding region comprises a functional pl9 promoter and encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide; b) the first AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity; and c) the AAV cap coding region comprises an AAV Cap intron and encodes a VP1, VP2 and VP3 polypeptide.

[Embodiment 58.] The recombinant cell of embodiment 57, wherein the second polynucleotide comprises in 5' to 3' order an eighth promoter operably linked to a first AAV Rep coding region, a ninth promoter operably linked to an AAV Cap coding region and a tenth promoter operably linked to a second AAV Rep coding region, wherein the second AAV Rep coding region encodes a Rep52 and Rep40 polypeptide, and wherein second AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity. [Embodiment 59.] The recombinant cell of embodiment 57 or embodiment 58, wherein the one or more of the first AAV Rep coding region, second AAV Rep coding region and AAV Cap coding region comprises a poly A signal.

[Embodiment 60.] Tire recombinant cell of any one of embodiment 57 to embodiment 58, wherein one or more of the eighth, ninth and tenth promoter is an inducible promoter.

[Embodiment 61 ] The recombinant cell of embodiment 60, wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 62.] The recombinant cell of any one of embodiment 1 to embodiment 61, wherein the AAV rep gene and the AAV cap gene have the same serotype.

[Embodiment 63.] Tire recombinant cell of any one of embodiment 1 to embodiment 61, wherein the AAV rep gene and the AAV cap gene have different serotypes. [Embodiment 64 ] The recombinant cell of any one of embodiment 1 to embodiment 63, wherein the AAV rep gene comprises an AAV2 rep gene.

[Embodiment 65.] The recombinant cell of any one of embodiment 1 to embodiment 64, wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. AAV11, AAV12, AAV13. AAV14, AAV15 and AAV16, AAV.rh8. AAV.rhlO. 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. [Embodiment 66.] Tire recombinant cell of embodiment 65, wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37.

[Embodiment 67.] The recombinant cell of embodiment 65, wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV8 or AAV9 serotype.

[Embodiment 68.] Hie recombinant cell of embodiment 65, wherein the AAV cap gene comprises the AAV2 serotype.

[Embodiment 69.] The recombinant cell of embodiment 65, wherein the AAV cap gene comprises the AAV6 serotype.

[Embodiment 70.] The recombinant cell of embodiment 65, wherein the AAV cap gene comprises the AAV8 serotype.

[Embodiment 71.] Hie recombinant cell of embodiment 65, wherein the AAV cap gene comprises the AAV9 serotype.

[Embodiment 72.] The recombinant cell of any one of embodiment 1 to embodiment 71 comprising the first polynucleotide.

[Embodiment 73.] The recombinant cell of any one of embodiment 1 to embodiment 71 comprising the second polynucleotide.

[Embodiment 74.] Hie recombinant cell of any one of embodiment 1 to embodiment 71 comprising the first and second polynucleotides.

[Embodiment 75.] The recombinant cell of embodiment 74, further comprising a sixth polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell.

[Embodiment 76.] The recombinant cell of embodiment 75, wherein the sixth polynucleotide is stably integrated.

[Embodiment 77.] Tire recombinant cell of any one of embodiment 1 to embodiment 76, wherein the cell is a HEK293 cell. HEK293 derived cell, CHO cell. CHO derived cell, HeLa cell. SF-9 cell, BHK cell, Vero cell, or PerC6 cell.

[Embodiment 78.] The recombinant cell of embodiment 77, wherein tire cell is a HEK293 cell or HEK293 derived cell.

[Embodiment 79.] Hie recombinant cell of any one of embodiments 1 to 78, wherein the first and second polynucleotides are comprised by a packaging polynucleotide.

[Embodiment 80. J The recombinant cell of embodiment 79, wherein the first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and the second polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-91.

[Embodiment 81.] The recombinant cell of embodiment 79, wherein the first polynucleotide comprises SEQ ID NO: 48 and the second polynucleotide comprises any one of SEQ ID NO: 70- 73 and 77-91.

[Embodiment 82.] The recombinant cell of embodiment 79, wherein the first polynucleotide comprises SEQ ID NO: 48 and the second polynucleotide comprises any one of SEQ ID NO: 70- 73 and 77-82.

[Embodiment 83.] The recombinant cell of any one of embodiments 79 to 82, wherein the packaging polynucleotide further comprises flanking recognition sites for an integrase or recombinase such that the packaging polynucleotide can be integrated into a genomic landing site of the host cell comprising the complementary recognition site.

[Embodiment 84 ] The recombinant cell of any one of embodiments 79 to 83, comprising a sixth polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell.

[Embodiment 85.] Tire recombinant cell of embodiment 84, wherein the sixth polynucleotide is stably integrated. [Embodiment 86 ] A recombinant cell comprising a stably integrated packaging polynucleotide comprising in a 5 ’to 3 ’ direction,

(1) an inducible promoter operably linked to

(2) a Rep cassette comprising, from 5’ to 3’, an AAV2 Rep78/68 coding region and a Rep52/40 coding region , and

(3) a CMV promoter operably linked to

(4) an AAV Cap coding region, operably linked to

(5) a Helper cassette comprising, from 5’ to 3’,

(a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP),

(b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide,

(c) an inducible promoter operably linked to a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide, optionally wherein the inducible promoter is a tetracycline inducible promoter, and

(d) a polynucleotide encoding an adenovirus VA RNA gene, wherein the 5' to 3' orientation of both tire polynucleotide encoding the E2A DBP and the polynucleotide encoding the VA RNA are opposite to the 5' to 3' orientation of tire adenovirus L4 22K/33K polynucleotide and the E4 orf polynucleotide.

[Embodiment 87.] The recombinant cell of embodiment 86, wherein the gene encoding the adenovirus L4 22K/33K polypeptide comprises an L4 22K/33K promoter operably linked to an L4 22K/33K coding region.

[Embodiment 88.] The recombinant cell of embodiment 86, wherein the gene encoding the adenovirus L4 22K/33K polypeptide comprises an inducible promoter operably linked to an L4 22K/33K coding region, optionally wherein the inducible promoter is a tetracycline inducible promoter.

[Embodiment 89 ] The recombinant cell of any one of embodiments 86 to 88, wherein the packaging polynucleotide further comprises flanking recognition sites for an integrase or recombinase such that tire packaging polynucleotide can be integrated into a genomic landing site of the host cell comprising the complementary recognition site.

[Embodiment 90.] Tire recombinant cel of any one of embodiments 86 to 88, wherein the cell further comprises a stably integrated polynucleotide comprising in a 5 ’to 3 ’ direction, a) a 5’ AAV2 inverted terminal repeat sequence (5’ 1TR), b) a promoter operably linked to a transgene coding region encoding a gene product, optionally wherein the promoter is a tissue-specific promoter to drive transgene expression in a target tissue cell, and c) a 3’ AAV2 inverted terminal repeat sequence (5’ ITR).

[Embodiment 91.] A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of embodiment 72: b) introducing into the cell i. a polynucleotide encoding an AAV capsid protein; ii. a polynucleotide encoding a functional rep gene; and iii. a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell, and c) maintaining the cell under conditions that allow production of the rAAV particles. [Embodiment 92.] A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of embodiment 73; b) introducing into the cell i. a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; ii. a polynucleotide comprising sufficient helper functions to permit packaging of the genome into the AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid, and c) maintaining the cell under conditions that allow production of the rAAV particles. [Embodiment 93.] A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of embodiment 74; b) introducing into the cell a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; and c) maintaining the cell under conditions that allow production of the rAAV particles. [Embodiment 94.] A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of embodiment 75 or embodiment 76; and b) maintaining the cell under conditions that allow production of tire rAAV particles. [Embodiment 95.] A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of any one of embodiments 84, 86 and 90; and b) maintaining the cell under conditions that allow production of the rAAV particles. [Embodiment 96.] The method of any one of embodiments 91 to 95, wherein the cell culture is a suspension culture.

[Embodiment 97.] Hie method of any one of embodiments 91 to 96, further comprising recovering the rAAV particles.

[Embodiment 98. J The method of any one of embodiments 91 to 97. wherein the cell culture has a volume between about 50 liters and about 20,000 liters.

[Embodiment 99.] The method of any one of embodiments 91 to 98, wherein the gene product is a polypeptide or a double stranded RNA molecule.

[Embodiment 100.] Hie method of embodiment 99. wherein the gene product is a polypeptide.

[Embodiment 101.] The method of embodiment 100, 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 non-mcmbranc associated splice variant ofVEGF receptor 1 (sFlt-1).

[Embodiment 102.] Hie method of embodiment 100, wherein the gene product is an gamma- sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65). cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated 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), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3). transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell line- derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatasc B (ARSB), N-acetyl-alpha- glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), betagalactosidase (GLB1). lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 90TC), survival motor neuron (SMN1), survival motor neuron (SMN2). neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF). mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4). protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgGl) Fc fusion. [Embodiment 103.] The method of embodiment 100, wherein the gene product is a dystrophin or a microdystrophin.

[Embodiment 104.] Tire method of embodiment 99. wherein the gene product is a microRNA, snRNA or an antisense RNA.

[0013] Still other features and advantages of the compositions and methods described herein wall become more apparent from the following detailed description when read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Functional evaluation of the p5 cis-regulatory element and split Rep/Cap plasmid constructs for rAAV8 production. (A, C) Schematic depiction of Rep/Cap plasmid constructs. Replication (Rep) and capsid (Cap) open reading frames are indicated. Arrow denotes a promoter, asterisk denotes sequence mutation, black circle denotes a poly A. All components are drawn to approximate scale. (B, D) HEK293 cells were triple transfected with each Rep/Cap plasmid, Helper (pAdDeltaF6-KanR) plasmid and transgene plasmid. rAAV8 crude viral genome (VG) titers were analyzed 72 h post-transfection, expressed as a relative percentage compared to the conventional Rep/Cap plasmid (B) or Rep/Cap 1.2 (D). Data shown are the mean ± S.D. of three independent biological replicates.

[0015] Figure 2. Functional evaluation of different Rep proteins within the split Rep/Cap plasmid construct for rAAV8 production. (A) Schematic depiction of Rep/Cap plasmid constructs. Replication (Rep) and capsid (Cap) open reading frames are indicated. Rep78 and Rcp52 w ere removed by deleting the alternate splice site within the rep codons to produce only Rep68 and Rep40 proteins. The Rep52/40 Kozak sequence was optimized by mutating TACATGG — > ATCATGG (start codon underlined). Arrow denotes a promoter, asterisk denotes sequence mutation, black circle denotes a poly A. All components are drawn to approximate scale. (B) HEK293 cells were triple transfected with each Rep/Cap plasmid, Helper (pAdDeltaF6- KanR) plasmid and transgene plasmid. rAAV8 crude viral genome (VG) titers were analyzed 72 h post-transfection, expressed as a relative percentage compared to the Rep/Cap 1.2. Data shown are the mean ± S.D. of three independent biological replicates.

[0016] Figure 3. Functional evaluation of the E2A and L4-33k/22k Helper components for rAAV8 production. (A) Schematic depiction of the E2A and L4-33k/22k open reading frames within Helper plasmid constructs (E4 and VA RNA are not indicated). A L4 promoter-driven plasmid expressing only the L4-33k/22k protein was also constructed (L4-33k/22k plasmid). Arrow denotes a promoter, black circle denotes a poly A. All components are drawn to approximate scale. (B) HEK293 cells were triple transfected with each Helper plasmid, Rep/Cap 1.2 plasmid and transgene plasmid. Hie L4-33k/22k plasmid was spiked at equal molar ratio to the E2Amin2 Helper plasmid. rAAV8 crude viral genome (VG) titers were analyzed 72 h posttransfection, expressed as a relative percentage compared to the Helper plasmid consisting the complete E2A and L4-33k/22k components. Data shown arc the mean ± S.D. of four independent biological replicates.

[0017] Figure 4. Functional evaluation of the E4 Helper components for rAAV8 production. (A) Schematic depiction of the E4 orfs (open reading frames) within the Helper plasmid constructs (E2A, L4-33k/22k and VA RNA are not indicated). Arrow denotes a promoter, black circle denotes a poly A. All components are drawn to approximate scale. (B) and (C) HEK293 cells were triple transfected with each Helper plasmid, Rep/Cap 1.2 plasmid and transgcnc plasmid. rAAV8 crude viral genome (VG) titers were analyzed 72 h post-transfection, expressed as a relative percentage compared to the full-length E4 gene. Data shown are tire mean ± S.D. of three independent biological replicates.

[0018] Figure 5. Evaluation of engineered Helper and Rep/Cap plasmid combinations for rAAV8 production. (A) Schematic depiction of the Helper plasmid constructs (VA RNA is not indicated). The E4orf6-6/7 Helper plasmid (Figure 4A) is denoted as Helper 2.0. Arrow denotes a promoter, black circle denotes a poly A. All components are drawn to approximate scale. (B) HEK293 cells were triple transfected with the Helper plasmid, Rep/Cap plasmid and transgene plasmid. rAAV8 crude viral genome (VG) titers were analyzed 72 h post-transfection, expressed as a relative percentage compared to tire Helper 2.0 and Rep/Cap 1.2 plasmid combination. [0019] Figure 6. Determination of product quality of the engineered Helper and Rep/Cap plasmids for rAAV8 production. The original pAdDeltaF6-KanR Helper plasmid is denoted as Helper 1.0. (A) Intact capsids were quantified at 72 h post-transfection using rAAV8-specific capsid ELISA and expressed as a relative percentage compared to the Helper 1.0 and Rep/Cap 1.2 plasmid combination. (B) The full/empty capsid ratio was calculated from the measured intact capsids in A and its VG titer. All data shown are tire mean ± S.D. of three independent biological replicates.

[0020] Figure 7. The Kozak sequence from Rep52/42 gene can be optimized to increase gene expression. 8 pg plasmid DNA encoding maxGFP, driven by the p!9 promoter with either the endogenous or optimized Kozak sequence (Table 4), were transfected into 1* 10⁷ HEK293 cells using PEImax as previously described. [Reference 24] A promoterless vector was used as a negative control. Cells were cultured in tube-spin bioreactors at 37°C and GFP fluorescence was measured at 48 h post-transfection as previously described. [Reference 24] Data are expressed as a fold-change with respect to the GFP fluorescence of the endogenous Kozak sequence. Data shown arc the mean ± S.D. of three independent biological replicates.

[0021] Figure 8. Endogenous AAV and the CMV promoters drive varying gene expression levels in non-AAV producing (S) and AAV producing (□; empty capsids) HEK293 cells. Vectors encoding maxGFP, driven by an endogenous AAV or the CMV promoter (Table 3), were co-transfected with Rep/Cap (pAAV2/8) and Helper (pAdDeltaF6-KanR) into 1 x 10⁷ HEK293 cells at 4: 1:2 weight ratio (GFP:pAAV2/8:pAdDeltaF6; 10 pg total DNA) using PEImax as previously described. [Reference 24] Rep/Cap and Helper plasmids with dclctcd/inactivatcd p5, p!9, p40, E2 -early and E4 promoters/introns (Tables 3 and 4) were used for the non-AAV producing condition. A promoterless vector was used as a negative control. Cells were cultured in tube-spin bioreactors at 37°C and GFP fluorescence was measured at 48 h post-transfection (A) and normalized against the CMV promoter (B) as previously described. [Reference 24] Data shown are the mean ± S.D. of three independent biological replicates..

[0022] Figure 9. Distribution of discrete transcription factor binding sites (TFBSs) within the E4, E2 -early and CMV promoters. Promoters were surveyed for the presence of discrete TFBSs using Genomatix Gene Regulation software as previously described. [Reference 24] Active transcription factors in HEK293 cells include E4F, JunB. Oct, AhR:ARNT, CREB, Spl, MYBL1, c-Rel, ZBED1 and NF-KB. [Reference 24] [0023] Figure 10. Schematic drawing of three plasmid transient transfection system for AAV production.

[0024] Figure 11. AAV Genome map.

[0025] Figure 12. Design of polynucleotides for inducible Rep/Cap expression. Arrows represent promoters (Tet: tetracycline inducible promoter: CMV : CMV promoter), boxes represent coding regions; IRES: internal ribosomal entry site.

[0026] Figure 13. Design of polynucleotides for inducible helper function expression. Arrows represent promoters (Tet: tetracycline inducible promoter; CMV: CMV promoter), boxes represent coding regions.

[0027] Figure 14. rAAV production in transiently transfected HEK293 derived cells using constitutively expressed helper construct and inducible Rep/Cap construct.

[0028] Figure 15. rAAV production in transiently transfected HEK293 derived cells using inducible helper construct and inducible Rep/Cap construct.

[0029] Figure 16. Design of polynucleotides for inducible helper function expression. Arrows represent promoters (Tet: tetracycline inducible promoter), boxes represent coding regions.

[0030] Figure 17. rAAV production in transiently transfected HEK293 derived cells using inducible helper construct and inducible Rep/Cap construct.

[0031] Figure 18. Design of polynucleotides for inducible Rep/Cap expression. Arrows represent promoters (Tet: tetracycline inducible promoter), boxes represent coding regions; IRES: internal ribosomal entry site.

[0032] Figure 19. rAAV production in transiently transfected HEK293 derived cells using inducible helper construct and inducible Rep/Cap construct.

[0033] Figure 20. Design of poly nucleotides for inducible Rep/Cap expression. Arrows represent promoters (Tet-on: tetracycline inducible promoter, CMV: CMV promoter), boxes represent coding regions; IRES: internal ribosomal entry site.

[0034] Figure 21. Design of polynucleotides for inducible helper function expression. Arrows represent promoters (TRE3G Bi promoter: tetracycline inducible bidirectional promoter), boxes represent coding regions.

[0035] Figure 22. Design of poly nucleotides for inducible Rep/Cap and helper function expression. Tet-on: tetracycline inducible promoter, TRE3G Bi promoter: tetracycline inducible bidirectional promoter), boxes represent coding regions. [0036] Figure 23. rAAV production in transiently transfected HEK293 derived cells using inducible helper construct and inducible Rep/Cap construct.

[0037] Figure 24. Design of polynucleotides for rAAV production by stably integrating a first packaging polynucleotide comprising trans (i.e.. Rep/Cap encoding) and helper sequences and a second cis polynucleotide encoding an rAAV genome into a safe harbor genomic site of a host cell (e.g., HEK293 cell). LHA and RHA represent BxBbl integrase integration sites/landing pads for insertion of multiple copies of transgene: TetON represents tetracycline inducible promoter; TetOn linked to RepCap represents a polynucleotide encoding inducible Rep and Cap genes; E2A represents a polynucleotide encoding an E2A coding region; L4 22K/33k promoter and L4 22K/33K represent a polynucleotide encoding an adenovirus L4 22K/33K promoter and coding region, respectively: E4 Orf6,7 represents a polynucleotide encoding the E4 ORF 6-6/7 coding region; VA1/11 RNA represents a polynucleotide encoding an adenovirus VA RNA gene; 5' ITR and 3' ITR represent AAV ITRs flanking the transgene of interest encoded by the rAAV; and Tissue Specific Promoter and Transgene represent polynucleotides encoding the gene of interest encoded by the rAAV. Orientation of arrow denotes the direction of transcription for promoters and coding regions.

[0038] Figure 25. Design of polynucleotides for rAAV production by stably integrating a first packaging polynucleotide comprising trans (i.e., Rep/Cap encoding) and helper sequences and a second cis polynucleotide encoding an rAAV genome into a safe harbor genomic site of a host cell (e.g., HEK293 cell). LHA and RHA represent BxBbl integrase integration sites/landing pads for insertion of multiple copies of transgcnc; TctON represents tetracycline inducible promoter; TetOn linked to RepCap represents a polynucleotide encoding inducible Rep and Cap genes; E2A represents a polynucleotide encoding an E2A coding region; L4 22K/33K represents a polynucleotide encoding an adenovirus L4 22K/33K coding region; E4 Orf6,7 represents a polynucleotide encoding the E4 ORF 6-6/7 coding region; VAI/II RNA represents a polynucleotide encoding an adenovirus VA RNA gene; 5' ITR and 3' ITR represent AAV ITRs flanking the transgene of interest encoded by the rAAV; and Tissue Specific Promoter and Transgene represent polynucleotides encoding tire gene of interest encoded by the rAAV. Orientation of arrow denotes tire direction of transcription for promoters and coding regions.

[0039] Figure 26. Design of polynucleotides for rAAV production by stably integrating a first packaging polynucleotide comprising trans (i.e., Rep/Cap encoding) and helper sequences and a second cis polynucleotide encoding an rAAV genome into a safe harbor genomic site of a host cell (e.g., HEK293 cell). LHA and RHA represent BxBbl integrase integration sites/landing pads for insertion of multiple copies of transgene; TetON represents tetracycline inducible promoter; TetOn linked to RepCap represents a polynucleotide encoding inducible Rep and Cap genes; E2A represents a polynucleotide encoding an E2A coding region; L4 22K/33k promoter and L4 22K/33K represent a polynucleotide encoding an adenovirus L4 22K/33K promoter and coding region, respectively; E4 Orf6,7 represents a polynucleotide encoding the E4 ORF 6-6/7 coding region; VAI/II RNA represents a polynucleotide encoding an adenovirus VA RNA gene; 5' ITR and 3' ITR represent AAV ITRs flanking the transgene of interest encoded by tire rAAV; and Tissue Specific Promoter and Transgene represent polynucleotides encoding the gene of interest encoded by the rAAV. Orientation of arrow denotes the direction of transcription for promoters and coding regions.

DETAILED DESCRIPTION

[0040] In one aspect, provided herein are recombinant polynucleotides, polynucleotide vectors and recombinant cells suitable for use in the production of recombinant AAV particles. Also provided herein are methods for producing rAAV particles.

DEFINITIONS

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. To facilitate an understanding of the disclosed methods, a number of tenns and phrases are defined below.

[0042] " AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or modifications, derivatives, or pseudotypes thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. 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 ovine AAV, and modifications, derivatives, or pseudotypes thereof. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0043] " Recombinant", as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is distinct from an AAV particle in nature.

[0044] A recombinant adeno-associated virus particle "rAAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector genome comprising a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). The rAAV particle may be of any AAV serotype, including any modification, derivative or pscudotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or derivatives/modifications/pseudotypes thereof). Such AAV serotypes and derivatives/modifications/pseudotypes, and methods of producing such serotypes/derivatives/modifications/ pscudotypcs are known in the art (see, e.g., Asokan et al., Mol. Thor. 20(4):699-708 (2012).

[0045] The rAAV particles of the disclosure may be of any seroty pe, or any combination of serotypes, (e.g., a population of rAAV particles that comprises two or more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles). In some embodiments, the rAAV particles are rAAVl, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV 10, or other rAAV particles, or combinations of two or more thereof). In some embodiments, the rAAV particles arc rAAV8 or rAAV9 particles.

[0046] 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 and AAV16 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype of AAV8, AAV9, or a derivative, modification, or pseudotype thereof.

[0047] The temi "cell culture," refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and tire like, as well as the components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large and small-scale production of proteins are encompassed by the present 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 stirred bioreactors. In some embodiments, the term "cell culture" refers to cells grown in a perfusion culture. In some embodiments, the term "cell culture" refers to cells grown in an alternating tangential flow (ATF) supported high-density perfusion culture.

[0048] The terms "purifying", "purification", "separate", "separating", "separation", "isolate", "isolating", or "isolation", as used herein, refer to increasing the degree of purity of a target product, e.g., rAAV particles and rAAV genome from a sample comprising the target product and one or more impurities. Typically, the degree of purity of the target product is increased by removing (completely or partially) at least one impurity from the sample. In some embodiments, the degree of purity of the rAAV in a sample is increased by removing (completely or partially) one or more impurities from the sample by using a method described herein.

[0049] "About" modifying, for example, the quantity of an ingredient in the compositions, concentration of an ingredient in the compositions, flow rate, rAAV particle yield, feed volume, salt concentration, and like values, and ranges thereof, employed in the methods provided herein, refers to variation in tire numerical quantity that can occur, for example, through typical measuring and handling procedures used for making concentrates or use solutions; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of a composition with a particular initial concentration or mixture. The term "about" also encompasses amounts that differ due to mixing or processing a composition with a particular initial concentration or mixture. Whether or not modified by the term "about" the claims include equivalents to the quantities. In some embodiments, the term "about" refers to ranges of approximately 10-20% greater than or less than the indicated number or range. In further embodiments, "about" refers to plus or minus 10% of the indicated number or range. For example, "about 10%" indicates a range of 9% to 11%. [0050] As used in the present disclosure and claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherw ise.

[0051] It is understood that wherever embodiments are described herein with tire language "comprising" otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of otherwise analogous embodiments described in terms of "consisting of are also provided.

[0052] Tire term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass 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).

[0053] Where embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, tire disclosed method encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also tire main group absent one or more of the group members. Tire disclosed methods also envisage the explicit exclusion of one or more of any of tire group members in the disclosed methods.

RECOMBINANT POLYNUCLEOTIDES

[0054] In some embodiments, the disclosure provides an isolated recombinant polynucleotide encoding one or more helper functions, one or more AAV rep genes, an AAV cap gene, or any combination thereof In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the one or more AAV rep genes encode a Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, Rep40 polypeptide or any combination thereof. In some embodiments, the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the AAV cap gene further encodes an assembly-activating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP). In some embodiments, the disclosure further provides a host cell comprising one or more recombinant polynucleotides described herein. In some embodiments, the one or more recombinant polynucleotides described herein are stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the host cell is aHEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the host cell is a EIEK293 cell or a HEK293 derived cell.

Polynucleotides encoding one or more helper functions

[0055] In some embodiments, the disclosure provides an isolated recombinant polynucleotide encoding one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), and an adenovirus VA RNA gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the recombinant polynucleotide encoding one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide.

[0056] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprise an inducible promoter. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the inducible promoter is a bidirectional promoter regulating the expression of the adenovirus E2A DNA binding protein (DBP) and the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide). In some embodiments, the inducible promoter is a tetracycline inducible promoter.

Adenovirus E2A DNA binding protein

[0057] In some embodiments, the adenovirus E2A DBP 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 to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8.

[0058] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a promoter operably linked to a polynucleotide encoding the E2A DNA binding protein (DBP). A skilled artisan understands that the gene expressing the adenovirus E2A DNA binding protein (DBP) can further comprise additional transcriptional control elements, such as an enhancer and a poly A signal. In some embodiments, the gene expressing tire adenovirus E2A DNA binding protein (DBP) comprises a poly A signal. A skilled artisan further understands that the polynucleotide encoding the E2A DNA binding protein (DBP) can encode, in addition to the protein coding regions, sequence elements that regulate the processing and/or translation of the E2A DBP RNA. In some embodiments, the polynucleotide encoding the E2A DNA binding protein (DBP) comprises one or more of a 5’ untranslated region, a coding region, a 3’ untranslated region, one or more exons, one or more introns, Kozak sequence, and an IRES (internal ribosomal entry site). In some embodiments, the polynucleotide encoding the E2A DNA binding protein (DBP) comprises an intron comprising a gene expressing an adenovirus L4 22K/33K polypeptide.

[0059] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a promoter operably linked to a polynucleotide encoding tire E2A DNA binding protein (DBP), wherein the promoter is an inducible promoter. In some embodiments, the inducible promoter is an alcohol inducible promoter, a forskolin inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter, a rapamycin inducible promoter, a hormone inducible promoter and a metal ion inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible 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 to SEQ ID NO: 1, 2 or 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, tire tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:

3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3.

[0060] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a promoter operably linked to a polynucleotide encoding the E2A DNA binding protein (DBP), wherein the promoter is a CMV promoter or an engineered CMV promoter or transcriptionally active fragment thereof. In some embodiments, the promoter is a CMV promoter. In some embodiments, the CMV 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 to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the promoter is an engineered CMV promoter or transcriptionally active fragment thereof. Engineered CMV immediate early promoters or transcriptionally active fragments thereof are known to one of skill, for example, as disclosed in International Publication No. WO2023141582A1. which is incorporated herein by reference in its entirety.

[0061] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a promoter operably linked to a polynucleotide encoding the E2A DNA binding protein (DBP), wherein the polynucleotide encoding the E2A DNA binding protein (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: 6 or 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6. In some embodiments, tire polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises SEQ ID NO: 7. In some embodiments, the adenovirus E2A DBP 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 to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises 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 to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a polyA signal.

[0062] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding an E2A DNA binding protein (DBP), wherein the tetracycline inducible 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’ to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises 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 to SEQ ID NO: 8. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding an E2A DNA binding protein (DBP), wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises 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 to SEQ ID NO: 8. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding an E2A DNA binding protein (DBP), wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises 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 to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, tire nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) further comprises a polyA signal. [0063] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (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: 9. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO:

9. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) further comprises a polyA signal. Adenovirus E4 polypeptide

[0064] In some embodiments, the gene expressing adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding the adenovirus E4 polypeptide. The adenovirus E4 region has the potential to transcribe and encode 7 different polypeptides that are resulted from differential splicing of a single primary transcript (orfl, 2. 3, 3/4, 4, 6. 6/7) generated by this promoter. The pattern of differential splicing for this transcript changes during the phases of viral infection with some appearing only in early phases and others in late phase (Dix et al., Journal of General Virology (1995), 76, 1051-1055). The encoded protein products of orfl, orf2, orf3, orf4, orf6, and orf6/7 were reported to exist in infected cells except for orf3/4, which might be absent or expressed below detection limit (Tauber et al., Gene 278 (2001) 1-23). orfl encoded protein is expressed in the late phase and targets a family of cellular proteins that play a role in cell signaling and signal transfection. There is no functional information about E4 product encoded by orf2. Furthermore, Ad5 mutants in which E4 orf2 were deleted, were about to grow to wild-type levels (Tauber et al., Gene 278 (2001) 1-23). In some embodiments, the E4 polypeptide comprises the E4 orfl -6/7, E4 orf2-6/7, E4 orf3-6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, or E4 orf3+6-6/7. E4 orf6/7 is also referenced as E4 orf7 in the art; E4 orf6/7 and E4 orf7 are used interchangeably herein. In some embodiments, the E4 polypeptide comprises the E4 orfl-6/7. In some embodiments, the E4 polypeptide comprises tire E4 orf2-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf3-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf4-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6. In some embodiments, the E4 polypeptide comprises the E4 orf3+6-6/7.

[0065] In some embodiments, E4 orfl, orf2, orf3, orf4. orf6, and orf6/7 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 to SEQ ID NO: 10, 1 1, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orfl, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95% to SEQ ID NO: 10, 11, 12. 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orfl. orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orfl, orf4, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. A skilled artisan understands that the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) can further comprise additional transcriptional control elements, such as an enhancer and a poly A signal. In some embodiments, the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprises a poly A signal. A skilled artisan further understands that the polynucleotide encoding the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) can encode, in addition to the protein coding regions, sequence elements that regulate the processing and/or translation of the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide). In some embodiments, the polynucleotide encoding the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprises one or more of a 5 ’ untranslated region, a coding region, a 3 ’ untranslated region, one or more exons, one or more introns. Kozak sequence, and an IRES (internal ribosomal entry site).

[0066] In some embodiments, the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprises a promoter operably linked to a polynucleotide encoding the E4 polypeptide (e.g.. an E4orf6 or E4orf6-6/7 polypeptide), wherein the promoter is an inducible promoter. In some embodiments, the inducible promoter is an alcohol inducible promoter, a forskolin inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter, a rapamycin inducible promoter, a hormone inducible promoter and a metal ion inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible 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 to SEQ ID NO: 1. 2 or 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, tire tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO:

3. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3.

[0067] In some embodiments, the gene expressing the adenovirus E4 polypeptide (e.g.. an E4orf6 or E4orf6-6/7 polypeptide) comprises a promoter operably linked to a polynucleotide encoding the E4 polypeptide (e.g.. an E4orf6 or E4orf6-6/7 polypeptide), wherein the promoter is a CMV promoter or an engineered CMV promoter or transcriptionally active fragment thereof. In some embodiments, the promoter is a CMV promoter. In some embodiments, the CMV 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 to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the promoter is an engineered CMV promoter or transcriptionally active fragment thereof.

Engineered CMV immediate early promoters or transcriptionally active fragments thereof are known to one of skill, for example, as disclosed in International Publication No. WO2023141582A1, which is incorporated herein by reference in its entirety.

[0068] E4orf6 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding an E4 orf6 polypeptide, wherein the polynucleotide encoding the E4 orf6 polypeptide 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: 16. In some embodiments, the polynucleotide encoding the E4 orf6 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 16. In some embodiments, the polynucleotide encoding the E4 orf6 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 16. In some embodiments, the polynucleotide encoding the E4 orf6 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 16. In some embodiments, the polynucleotide encoding the E4 orf6 polypeptide comprises SEQ ID NO: 16. In some embodiments, the 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 to SEQ ID NO: 14. In some embodiments, the E4 orf6 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14. In some embodiments, the E4 orf6 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14. In some embodiments, the E4 orf6 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 14. In some embodiments, the E4 orf6 polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises 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 to SEQ ID NO: 14. In some embodiments, tire nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a poly A signal.

[0069] In some embodiments, the gene expressing the E4 orf6 polypeptide comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding an E4 orf6 polypeptide, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding an E4 orf6 polypeptide comprises 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 to SEQ ID NO: 14. In some embodiments, tire gene expressing the E4 orf6 polypeptide comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding an E4 orf6 polypeptide, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding an E4 orf6 polypeptide comprises 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 to SEQ ID NO: 14. In some embodiments, the gene expressing the E4 orf6 polypeptide comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding an E4 orf6 polypeptide, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein tire nucleotide sequence encoding an E4 orf6 polypeptide comprises 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 to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding an E4 orf6 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the gene expressing tire E4 orf6 polypeptide further comprises a polyA signal.

[0070] In some embodiments, the gene expressing the E4 orf6 polypeptide 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: 23. In some embodiments, the gene expressing the E4 orf6 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 23. In some embodiments, the gene expressing the E4 orf6 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 23. In some embodiments, the gene expressing tire E4 orf6 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 23. In some embodiments, tire gene expressing tire E4 orf6 polypeptide comprises the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the gene expressing the E4 orf6 polypeptide further comprises a polyA signal.

[0071] E4orfl-6/7 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding E4 orf 1 -6/7 polypeptides, wherein the polynucleotide encoding the E4 orf 1-6/7 polypeptides 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: 17. In some embodiments, the polynucleotide encoding the E4 orf 1-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 17. In some embodiments, the polynucleotide encoding the E4 orfl -6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 17. In some embodiments, the polynucleotide encoding the E4 orfl -6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 17. In some embodiments, the polynucleotide encoding the E4 orfl -6/7 polypeptides comprises SEQ ID NO: 17. In some embodiments, the E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprise 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orfl, 2, 3, 4. 6 and 6/7 polypeptides comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprise the amino acid sequence of SEQ ID NO: 10, 11. 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2. 3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3. 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 11. 12. 13. 14, and 15, respectively. In some embodiments, tire nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a polyA signal.

[0072] In some embodiments, the gene expressing the E4 orfl -6/7 polypeptides comprises a tetracy cline inducible promoter operably linked to a polynucleotide encoding E4 orfl. 2, 3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 poly peptides comprises 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 to SEQ ID NO: 10. 11, 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orfl -6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orfl -6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 10, 11, 12, 13, 14. and 15. respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfl, 2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 11. 12. 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orfl -6/7 polypeptides further comprises a polyA signal.

[0073] In some embodiments, the gene expressing the E4 orfl -6/7 polypeptides 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: 24. In some embodiments, the gene expressing the E4 orfl -6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 24. In some embodiments, the gene expressing the E4 orfl-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 24. In some embodiments, the gene expressing the E4 orfl-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 24. In some embodiments, the gene expressing the E4 orfl-6/7 polypeptides comprises the nucleotide sequence of SEQ ID NO: 24. In some embodiments, the gene expressing the E4 orfl-6/7 polypeptides further comprises a polyA signal. [0074] E4orf2-6/7 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding E4 orf2-6/7 polypeptides, wherein the polynucleotide encoding the E4 orf2-6/7 polypeptides 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: 18. In some embodiments, the polynucleotide encoding the E4 orf2-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the E4 orf2-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the E4 orf2-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the E4 orf2-6/7 polypeptides comprises SEQ ID NO: 18. In some embodiments, the E4 orf2, 3, 4, 6 and 6/7 polypeptides comprise 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 to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf2, 3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf2, 3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf2, 3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf2, 3, 4, 6 and 6/7 polypeptides comprise the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4. 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3. 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 11, 12, 13. 14. and 15. respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a polyA signal.

[0075] In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf2. 3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 11. 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 11, 12, 13. 14. and 15. respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4. 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf2, 3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides further comprises a polyA signal.

[0076] In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides 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: 25. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 25. In some embodiments, tire gene expressing the E4 orf2-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 25. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 25. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides comprises the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the gene expressing the E4 orf2-6/7 polypeptides further comprises a polyA signal.

[0077] E4orf3-6/7 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding E4 orf3-6/7 polypeptides, wherein the polynucleotide encoding the E4 orf3-6/7 polypeptides 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: 19. In some embodiments, the polynucleotide encoding tire E4 orf3-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the E4 orf3-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the E4 orf3-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the E4 orf3-6/7 polypeptides comprises SEQ ID NO: 19. In some embodiments, the E4 orf , 4, 6 and 6/7 polypeptides comprise 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 to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 12. 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise the amino acid sequence of SEQ ID NO: 12. 13. 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 or£3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 or£3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a poly A signal.

[0078] In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orfB, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 13, 14. and 15. respectively. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein tire nucleotide sequence encoding E4 orf3, 4. 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides further comprises a polyA signal. [0079] In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides 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: 26. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 26. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 26. In some embodiments, the gene expressing the E4 or£3-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 26. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides comprises the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the gene expressing the E4 orf3-6/7 polypeptides further comprises a polyA signal .

[0080] E4orf4-6/7 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding E4 orf4-6/7 polypeptides, wherein the polynucleotide encoding the E4 orf4-6/7 polypeptides 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: 20. In some embodiments, the polynucleotide encoding the E4 orf4-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the E4 orf4-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the E4 orf4-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the E4 orf4-6/7 polypeptides comprises SEQ ID NO: 20. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise 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 to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the E4 orf3, 4, 6 and 6/7 polypeptides comprise tire amino acid sequence of SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3. 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a polyA signal.

[0081] In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 poly peptides comprises 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 to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3. 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3, 4, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4. 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 4, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides further comprises a polyA signal.

[0082] In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides 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: 27. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 27. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 27. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 27. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides comprises the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the gene expressing the E4 orf4-6/7 polypeptides further comprises a polyA signal.

[0083] E4orf6-6/7 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding E4 orf6-6/7 polypeptides, wherein the polynucleotide encoding the E4 orf6-6/7 polypeptides 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: 21. In some embodiments, the polynucleotide encoding the E4 orf6-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 21. In some embodiments, the polynucleotide encoding the E4 orf6-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 21. In some embodiments, the polynucleotide encoding the E4 orf6-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 21. In some embodiments, the polynucleotide encoding the E4 orf6-6/7 polypeptides comprises SEQ ID NO: 21 . In some embodiments, the E4 orf6 and 6/7 polypeptides comprise 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 to SEQ ID NO: 14, and 15, respectively. In some embodiments, the E4 orf6 and 6/7 polypeptides comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the E4 orf6 and 6/7 polypeptides comprise an amino acid sequence having at least 95% identity' to SEQ ID NO: 14, and 15, respectively. In some embodiments, the E4 orf6 and 6/7 polypeptides comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the E4 orf6 and 6/7 polypeptides comprise the amino acid sequence of SEQ ID NO: 14, and 15, respectively . In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides is operably linked to a promoter (e g., tetracycline inducible promoter) and to a polyA signal.

[0084] In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf6 and 6/7 polypeptides, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides further comprises a polyA signal.

[0085] In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides 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: 28. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 28. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 28. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 28. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides comprises the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the gene expressing the E4 orf6-6/7 polypeptides further comprises a polyA signal.

[0086] E4orf3+6-6/7 In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a promoter operably linked to a polynucleotide encoding E4 orf3+6-6/7 polypeptides, wherein the polynucleotide encoding the E4 orf3+6-6/7 polypeptides 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. In some embodiments, the polynucleotide encoding the E4 orf3+6-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 22. In some embodiments, the polynucleotide encoding the E4 orf3+6- 6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 22. In some embodiments, the polynucleotide encoding the E4 orf3+6-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 22. In some embodiments, the polynucleotide encoding the E4 orf3+6-6/7 polypeptides comprises SEQ ID NO: 22. In some embodiments, the E4 orf3, 6 and 6/7 polypeptides comprise 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 to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the E4 orf3, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 12. 14, and 15, respectively. In some embodiments, the E4 orf3, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the E4 orf3, 6 and 6/7 polypeptides comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the E4 orfS, 6 and 6/7 polypeptides comprise the amino acid sequence of SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identify to SEQ ID NO: 12, 14. and 15. respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orfB, 6 and 6/7 polypeptides is operably linked to a promoter (e.g., tetracycline inducible promoter) and to a polyA signal.

[0087] In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3. 6 and 6/7 polypeptides, wherein the tetracycline inducible 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 to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orf3. 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises a nucleotide sequence having at least at least 95% identity to SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises a tetracycline inducible promoter operably linked to a polynucleotide encoding E4 orfi, 6 and 6/7 polypeptides, wherein the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1 or 2 and wherein the nucleotide sequence encoding E4 orf3. 6 and 6/7 polypeptides comprises 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 to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 14. and 15. respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the nucleotide sequence encoding E4 orf3, 6 and 6/7 polypeptides comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 14, and 15, respectively. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides further comprises a poly A signal. [0088] In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides 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: 29. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 29. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 29. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 29. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides comprises the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the gene expressing the E4 orf3+6-6/7 polypeptides further comprises a poly A signal.

Adenovirus VA RNA

[0089] In some embodiments, an adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30.

[0090] In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I 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: 31. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I comprising a nucleotide sequence having at least 90 % identity to SEQ ID NO: 31 . In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I comprising a nucleotide sequence having at least 95 % identity to SEQ ID NO: 31. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 31. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I comprising the nucleotide sequence of SEQ ID NO: 31. [0091] In some embodiments, the adenovirus VA RNA gene encodes a VA RNA II 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: 32. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA II comprising a nucleotide sequence having at least 90 % identity to SEQ ID NO: 32. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA II comprising a nucleotide sequence having at least 95 % identity to SEQ ID NO: 32. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA II comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 32. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA II comprising the nucleotide sequence of SEQ ID NO: 32.

[0092] In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I and II 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: 31 and 32, respectively. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I and 11 comprising a nucleotide sequence having at least 90 % identity to SEQ ID NO: 31 and 32, respectively. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I and II comprising a nucleotide sequence having at least 95 % identity to SEQ ID NO: 31 and 32, respectively. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I and II comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 31 and 32, respectively. In some embodiments, the adenovirus VA RNA gene encodes a VA RNA I and II comprising the nucleotide sequence of SEQ ID NO: 31 and 32, respectively.

Helper polynucleotides

[0093] In some embodiments, the disclosure provides an isolated recombinant polynucleotide encoding one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (c.g., an E4orf6 or E4orf6-6/7 polypeptide), and an adenovirus VA RNA gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the recombinant polynucleotide encoding one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the recombinant polynucleotide encoding one or more helper functions described herein comprises one or more additional sequence elements selected from poly A signal, Kozak sequence, insulator element (e.g., cHS4 element), and selectable marker gene.

[0094] In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprise an inducible promoter. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the inducible promoter is a bidirectional promoter regulating the expression of the adenovirus E2A DNA binding protein (DBP) and the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide). In some embodiments, the inducible promoter is a tetracycline inducible promoter.

[0095] In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises a (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide); (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises a, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide); (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP): and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the isolated recombinant polynucleotide comprises, in a 5' to 3' order, (a) cHS4 element, (b) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide); (c) cHS4 element, (d) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (e) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the first promoter, the second promoter, and the adenovirus VA RNA gene have the same 5' to 3' orientation. In some embodiments, the E4 polypeptide comprises the E4 orfl- 6/7, E4 orf2-6/7, E4 orf3-6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, or E4 orf3+6-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf 1-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf2-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf -6/7. In some embodiments, the E4 polypeptide comprises the E4 orf4-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6. In some embodiments, the E4 polypeptide comprises the E4 orf3+6-6/7. In some embodiments, E4 orfl, orf2, orf3, orf4. orf6, and orf6/7 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2. orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95%to SEQ ID NO: 10. 11. 12, 13, 14, and 15, respectively. In some embodiments. E4 orfl , orf2, orfl, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orfl, orf4, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the adenovirus E2A DBP 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 to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first promoter or second promoter comprises an inducible promoter. In some embodiments, the first promoter and second promoter comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter 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: 1 or 2. In some embodiments, the inducible promoter is a tetracycline inducible promoter comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 1 or 2. In some embodiments, the inducible promoter is a tetracycline inducible promoter comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 1 or 2. In some embodiments, the inducible promoter is a tetracycline inducible promoter comprising a nucleotide sequence having at least 98% identity to SEQ ID NO: 1 or 2. In some embodiments, the inducible promoter is a tetracycline inducible promoter comprising the nucleotide sequence of SEQ ID NO: 1 or 2. In some embodiment, the polynucleotide encoding an adenovirus E4 polypeptide (e.g , an E4orf6 or E4orf6-6/7 polypeptide) and the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) further comprise a poly A signal. In some embodiments, the poly A signal comprises the nucleotide sequence of SEQ ID NO: 42 or 43. In some embodiments, an adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the cHS4 element comprises the nucleotide sequence of SEQ ID NO: 40 or 41.

[0096] Original iHelper In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably- linked to a polynucleotide encoding an adenovirus E4 orf6 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf6 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, tire first and second promoters are tetracycline inducible promoters 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: 1 or 2, the E4 orf6 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 to SEQ ID NO: 14, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 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 to SEQ ID NO: 14, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 14, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 comprises the amino acid sequence of SEQ ID NO: 14, respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and tire polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6 polypeptide 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: 16, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6 polypeptide 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: 16, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 16, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 16, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 16, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7. and the polynucleotide encoding tire adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6 polypeptide comprises the nucleotide sequence of SEQ ID NO: 16, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal. In some embodiments, an expression cassette comprising the Original Helper genes is integrated into a safe harbor locus of a cell.

[0097] iHelper#l In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide: (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the E4 orf6 and orf6/7 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 to SEQ ID NO: 14 and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 and orf6/7 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 to SEQ ID NO: 14 and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 and orf6/7 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14 and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 and orf6/7 comprises an amino acid sequence having at least 95% identity’ to SEQ ID NO: 14 and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 and orf6/7 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 14 and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, and tire polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf6 and orf6/7 comprises the amino acid sequence of SEQ ID NO: 14 and 15, respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide 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: 21, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters arc tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, die polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide 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: 21, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding tire adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 21, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 21, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 21, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide comprises the nucleotide sequence of SEQ ID NO: 21, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7. and tire polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal. [0098] iHelper#2 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: I or 2, the E4 orf4, orf6, and orf6/7 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 to SEQ ID NO: 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8. and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf4, orf6, and orf6/7 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 to SEQ ID NO: 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, tire E4 orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and tire polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf4, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 13, 14. and 15. respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide 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: 20, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide 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: 20, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding tire adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, tire first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 20, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 20, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2. the polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 20, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, and tire polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf4-6/7 polypeptide comprises the nucleotide sequence of SEQ ID NO: 20, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal. [0099] iHelper#3 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide: (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the E4 orf3, orf4. orf6, and orf6/7 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 to SEQ ID NO: 12, 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf4, orf6, and orf6/7 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 to SEQ ID NO: 12, 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf4, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8. and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, tire polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide 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: 19, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide 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: 19, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 or£3-6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 19, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 19, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters arc tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 19, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3-6/7 polypeptide comprises the nucleotide sequence of SEQ ID NO: 19, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal.

[00100] iHelper#4 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the E4 orf2, orf3, orf4, orf6, and orf6/7 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 to SEQ ID NO: 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf2, orf3, orf4, orf6, and orf6/7 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 to SEQ ID NO: 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively, tire adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively, tire adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf2, orf3, orf4, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide 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: 18, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide 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: 18, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 18, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 18, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 18, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity' to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf2-6/7 polypeptide comprises the nucleotide sequence of SEQ ID NO: 18, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7. and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal.

[00101] iHelpe r#5 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf 1-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP): and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orfl-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP): and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 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 to SEQ ID NO: 10, 11. 12. 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2. the E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the polynucleotide encoding an adenovirus E4 orfl-6/7 polypeptide 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: 17, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orfl-6/7 polypeptide 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: 17, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orfl- 6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 17, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7. and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orfl-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 17, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orfl-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 17, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7. and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orfl-6/7 polypeptide comprises the nucleotide sequence of SEQ ID NO: 17, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal.

[00102] iHelpe r#7 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a first promoter operably linked to a polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide; (b) a second promoter operably linked to a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); and (c) a polynucleotide encoding the adenovirus VA RNA gene. In some embodiments, tire first and second promoters are tetracycline inducible promoters 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: 1 or 2, the E4 orf3, orf6, and orf6/7 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 to SEQ ID NO: 12, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, tire first and second promoters are tetracycline inducible promoters comprising tire nucleotide sequence of SEQ ID NO: 1 or 2, tire E4 orf3, orf6, and orf6/7 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 to SEQ ID NO: 12, 14, and 15, respectively, the adenovirus E2A DBP 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 to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf6, and orf6/7 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf6, and orf6/7 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf6, and orf6/7 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 12, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the E4 orf3, orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 12, 14, and 15, respectively, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters 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: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide 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, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide 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, the a polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, and the polynucleotide encoding tire adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 22, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 22, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2. the polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 22, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the first and second promoters are tetracycline inducible promoters comprising the nucleotide sequence of SEQ ID NO: 1 or 2, the polynucleotide encoding an adenovirus E4 orf3+6-6/7 polypeptide comprises the nucleotide sequence of SEQ ID NO: 22, the a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 6 or 7, and the polynucleotide encoding the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding tire E4 polypeptide and the polynucleotide encoding the E2A DBP further comprises a polyA signal. [00103] In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the isolated recombinant polynucleotide comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the isolated recombinant polynucleotide comprises, in a 5' to 3' order, a cHS4 element, a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), a cHS4 element, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), the gene expressing an adenovirus E2A DNA binding protein (DBP), and the adenovirus VA RNA gene have the same 5' to 3' orientation. In some embodiments, the E4 polypeptide comprises the E4 orf 1-6/7, E4 orf2-6/7, E4 orf3-6/7, E4 orf4- 6/7, E4 orf6-6/7, E4 orf6, or E4 orf3+6-6/7. E4 orf6/7 is also referenced as E4 orf7 in the art; E4 orf6/7 and E4 orf7 are used interchangeably herein. In some embodiments, the E4 polypeptide comprises the E4 orfl-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf2-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf3-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf4-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6. In some embodiments, the E4 polypeptide comprises tire E4 orf3+6-6/7. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the cHS4 element comprises the nucleotide sequence of SEQ ID NO: 40 or 41.

[00104] Original iHelper In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf6 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, the inducible promoters arc the same. In some embodiments, the inducible promoters arc different. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the gene expressing the adenovirus E4 polypeptide 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: 23; the gene expressing the adenovirus E2A DNA binding protein (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: 9; and tire gene expressing the adenovirus VA RNA 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: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 23; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 23; tire gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 23; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and tire gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 23; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, the recombinant polynucleotide 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: 33. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 33. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 33. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 33. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the recombinant polynucleotide 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: 93. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 93. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 93. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 93. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 93. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 93 is integrated into a safe harbor locus of a cell.

[00105] iHelper#5 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf 1-6/7 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the gene expressing the adenovirus E4 polypeptide 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: 24; tire gene expressing the adenovirus E2A DNA binding protein (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: 9; and the gene expressing the adenovirus VA RNA 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: 30. In some embodiments, tire gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 24; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 24; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 24; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 24; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, the recombinant polynucleotide 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: 34. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 34. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 34. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 34. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the recombinant polynucleotide 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: 94. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 94. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 94. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 94. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 94. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 94 is integrated into a safe harbor locus of a cell.

[00106] iHelper#4 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf2-6/7 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, tire inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, tire inducible promoter is a tetracycline inducible promoter. In some embodiments, the gene expressing the adenovirus E4 polypeptide 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: 25; the gene expressing the adenovirus E2A DNA binding protein (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: 9; and the gene expressing the adenovirus VA RNA 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: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 25; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 25; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 25; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 25; the gene expressing tire adenovirus E2A DNA binding protein (DBP) comprises tire nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, the recombinant polynucleotide 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: 35. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 35. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 35. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 35. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the recombinant polynucleotide 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: 95. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 95. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 95. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 95. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 95. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 95 is integrated into a safe harbor locus of a cell.

[00107] iHelper#3 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf3-6/7 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the gene expressing the adenovirus E4 polypeptide 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: 26; the gene expressing the adenovirus E2A DNA binding protein (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: 9; and the gene expressing the adenovirus VA RNA 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: 30. In some embodiments, tire gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 26; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 26; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 26; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 26; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, the recombinant polynucleotide 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: 36. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 36. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 36. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 36. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 36. In some embodiments, the recombinant polynucleotide 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: 96. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 96. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 96. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 96. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 96. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 96 is integrated into a safe harbor locus of a cell.

[00108] iHelpe r#2 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf4-6/7 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, tire inducible promoters are the same. In some embodiments, the inducible promoters arc different. In some embodiments, tire gene expressing the adenovirus E4 polypeptide 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: 27; the gene expressing the adenovirus E2A DNA binding protein (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: 9; and the gene expressing the adenovirus VA RNA 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: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 27; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 27; tire gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 27; the gene expressing tire adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and tire gene expressing tire adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 27; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, the recombinant polynucleotide 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: 37. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 37. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 37. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 37. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 37. In some embodiments, the recombinant polynucleotide 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: 97. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 97. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 97. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 97. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 97. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 97 is integrated into a safe harbor locus of a cell.

[00109] iHelper#l In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf6-6/7 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, the inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, the gene expressing the adenovirus E4 polypeptide 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: 28; the gene expressing the adenovirus E2A DNA binding protein (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: 9; and the gene expressing the adenovirus VA RNA 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: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 28; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 28; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, tire gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 28; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 28; tire gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, the recombinant polynucleotide 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: 38. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 38. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 38. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 38. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the recombinant polynucleotide 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: 98. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 98. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 98. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 98. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 98. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 98 is integrated into a safe harbor locus of a cell.

[00110] iHelper#7 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, a gene expressing an adenovirus E4 orf3+6-6/7 polypeptide, a gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and the gene expressing the adenovirus E4 polypeptide comprise an inducible promoter. In some embodiments, tire inducible promoters are the same. In some embodiments, the inducible promoters are different. In some embodiments, tire gene expressing tire adenovirus E4 polypeptide 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: 29; the gene expressing the adenovirus E2A DNA binding protein (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: 9; and the gene expressing the adenovirus VA RNA 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: 30. In some embodiments, tire gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 29; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 29; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 29; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the gene expressing the adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 29; the gene expressing the adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9; and the gene expressing the adenovirus VA RNA comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the gene expressing the E4 polypeptide and the gene expressing the E2A DNA binding protein (DBP) further comprise a polyA signal. In some embodiments, tire recombinant polynucleotide 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: 39. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 39. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 39. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 39. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 39. In some embodiments, the recombinant polynucleotide 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: 99. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 99. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 99. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 99. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 99. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 99 is integrated into a safe harbor locus of a cell.

[00111] iHelper#6 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises a (a) a bidirectional tetracycline inducible promoter operably linked to a polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) and a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP); (b) a gene encoding an adenovirus L4 22K/33K polypeptide, and (c) a polynucleotide encoding an adenovirus VA RNA gene. In some embodiments, the gene encoding an adenovirus L4 22K/33K polypeptide is located between the bidirectional tetracycline inducible promoter and the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP). In some embodiments, the recombinant polynucleotide comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a bidirectional tetracycline inducible promoter, (d) a polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), and (e) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), and wherein the 5' to 3' orientation of tire gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, tire adenovirus E2A DNA binding protein (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 to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the adenovirus E2A DBP polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide encoding the E2A DNA binding protein (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: 6 or 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6. In some embodiments, tire polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the adenovirus E2A DBP comprises SEQ ID NO: 7. In some embodiments, the adenovirus L4 22K/33K 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 to SEQ ID NO: 44. In some embodiments, the adenovirus L4 22K/33K polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 44. In some embodiments, adenovirus L4 22K/33K polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 44. In some embodiments, adenovirus L4 22K/33K polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 44. In some embodiments, the adenovirus L4 22K/33K polypeptide comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the gene encoding an adenovirus L4 22K/33K polypeptide comprises a promoter operably linked to a polynucleotide encoding the adenovirus L4 22K/33K polypeptide, wherein the 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 to SEQ ID NO: 45. In some embodiments, the L4 22K/33K promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 45. In some embodiments, the L4 22K/33K promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 45. In some embodiments, the L4 22K/33K promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 45. In some embodiments, the L4 22K/33K promoter comprises the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the gene encoding an adenovirus L4 22K/33K polypeptide comprises a promoter operably linked to a polynucleotide encoding the adenovirus L4 22K/33K polypeptide, wherein the polynucleotide encoding the adenovirus L4 22K/33K polypeptide 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: 46. In some embodiments, the polynucleotide encoding the adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 46. In some embodiments, the polynucleotide encoding the adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 46. In some embodiments, the polynucleotide encoding the adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 46. In some embodiments, the polynucleotide encoding the adenovirus L4 22K/33K polypeptide comprises the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the gene encoding an adenovirus L4 22K/33K polypeptide 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: 47. In some embodiments, the gene encoding the adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 47. In some embodiments, the gene encoding the adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 47. In some embodiments, the gene encoding the adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 47. In some embodiments, the gene encoding tire adenovirus L4 22K/33K polypeptide comprises the nucleotide sequence of SEQ ID NO: 47. In some embodiments, the bidirectional tetracycline inducible 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 to SEQ ID NO: 3. In some embodiments, the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3. In some embodiments, the bidirectional tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, an expression cassette comprising a iHelper#6 polynucleotide is integrated into a safe harbor locus of a cell.

[00112] In some embodiments, the E4 polypeptide comprises the E4 orfl-6/7, E4 orf2-6/7, E4 orfl-6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, or E4 orf3+6-6/7. In some embodiments, the E4 polypeptide comprises the E4 orfl-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf2-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf3-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf4-6/7. In some embodiments, tire E4 polypeptide comprises the E4 orf6-6/7. In some embodiments, the E4 polypeptide comprises the E4 orf6. In some embodiments, the E4 polypeptide comprises the E4 orf3+6-6/7. In some embodiments, E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 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 to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments. E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 90% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orfl, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 95% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, E4 orfl, orf2, orf3, orf4, orf6, and orf6/7 comprises an amino acid sequence having at least 98% to SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments. E4 orfl, orf2, orfl, orf , orf6, and orf6/7 comprises the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, a polynucleotide encoding E4 orfl-6/7, E4 orf2-6/7, E4 orfl-6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, and E4 orf3+6-6/7 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: 17, 18, 19, 20, 21, 16, and 22, respectively. In some embodiments, the polynucleotide encoding the adenovirus E4 orfl-6/7, E4 orf2-6/7, E4 orfl-6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, and E4 orf3+6-6/7 comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 17, 18, 19, 20, 21, 16, and 22, respectively. In some embodiments, the polynucleotide encoding the adenovirus E4 orf 1-6/7, E4 orf2-6/7, E4 orf? -6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, and E4 orf?+6-6/7 comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 17, 18, 19, 20, 21, 16, and 22, respectively. In some embodiments, the polynucleotide encoding the adenovirus E4 orf 1-6/7, E4 orf2-6/7, E4 orf? -6/7, E4 orf4-6/7, E4 orf6-6/7, E4 orf6, and E4 orf3+6-6/7 comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 17, 18, 19, 20, 21, 16. and 22. respectively. In some embodiments, the polynucleotide encoding the adenovirus E4 orf 1-6/7, E4 orf2-6/7, E4 orf? -6/7. E4 orf4-6/7, E4 orf6-6/7, E4 orf6, and E4 orf3+6-6/7 comprises the nucleotide sequence of SEQ ID NO: 17, 18, 19, 20, 21, 16, and 22, respectively. In some embodiments, an adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, an adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiment, the polynucleotide encoding an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) and the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) further comprise a poly A signal. In some embodiments, the poly A signal comprises the nucleotide sequence of SEQ ID NO: 42 or 43.

[00113] In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP). (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a bidirectional tetracycline inducible promoter, (d) a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide, and (e) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4orf6-6/7 polypeptide, and wherein the 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, (a) the adenovirus E2A DNA binding protein (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 to SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide 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: 47, (c) tire bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the E4 orf6 and orf6/7 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 to SEQ ID NO: 14 and 15, respectively, and (e) the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the E4 orf6 and orf6/7 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14 and 15, respectively, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the E4 orf6 and orf6/7 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14 and 15, respectively, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3,(d) tire E4 orf6 and orf6/7 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 14 and 15, respectively, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises the amino acid sequence of SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises the nucleotide sequence of SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3,(d) the E4 orf6 and orf6/7 polypeptide comprises the amino acid sequence of SEQ ID NO: 14 and 15. respectively, and (e) the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30.

[00114] In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a bidirectional tetracycline inducible promoter, (d) a polynucleotide encoding an adenovirus E4 orf6 polypeptide, and (e) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4orf6 polypeptide, and wherein the 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, (a) the adenovirus E2A DNA binding protein (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 to SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide 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: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the 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 to SEQ ID NO: 14, and (e) the adenovirus VA RNA 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’ to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the E4 orf6 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the E4 orf6 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3,(d) the E4 orf6 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 14, and (e) tire adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, (a) the adenovirus E2A DNA binding protein (DBP) comprises the amino acid sequence of SEQ ID NO: 8, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises the nucleotide sequence of SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3 ,(d) the E4 orf6 polypeptide comprises the amino acid sequence of SEQ ID NO: 14, and (e) the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30.

[00115] In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a bidirectional tetracycline inducible promoter, (d) a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide, and (e) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4orf6-6/7 polypeptide, and wherein the 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide 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: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) tire polynucleotide encoding E4orf6-6/7 polypeptide 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: 21, and (e) the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the Polynucleotide encoding E4orf6-6/7 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 21, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7. (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the Polynucleotide encoding E4orf6-6/7 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 21, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity’ to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3,(d) the Polynucleotide encoding E4orf6-6/7 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 21, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity- to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the amino acid sequence of SEQ ID NO: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises the nucleotide sequence of SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3,(d) the Polynucleotide encoding E4orf6-6/7 polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and (e) the adenovirus VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30.

[00116] In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a bidirectional tetracycline inducible promoter, (d) a polynucleotide encoding an adenovirus E4 orf6 polypeptide, and (e) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4orf6 polypeptide, and wherein the 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, (a) tire polynucleotide encoding an adenovirus E2A DNA binding protein (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: 6 or 7, (b) tire gene encoding an adenovirus L4 22K/33K polypeptide 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: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the polynucleotide encoding E4orf6 polypeptide 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: 14, respectively, and (e) the adenovirus VA RNA 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 to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) the polynucleotide encoding E4orf6 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 16, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6 or 7. (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3,(d) tire polynucleotide encoding E4orf6 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 16, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3,(d) the polynucleotide encoding E4orf6 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 16, and (e) the adenovirus VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, (a) the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises the amino acid sequence of SEQ ID NO: 6 or 7, (b) the gene encoding an adenovirus L4 22K/33K polypeptide comprises the nucleotide sequence of SEQ ID NO: 47, (c) the bidirectional tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3,(d) the polynucleotide encoding E4orf6 polypeptide comprises the amino acid sequence of SEQ ID NO: 16, and (e) the adenovirus VA RNA gene comprises tire nucleotide sequence of SEQ ID NO: 30. [00117] iHelper#6 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a bidirectional tetracycline inducible promoter, (d) a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide, and (e) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4orf6-6/7 polypeptide and wherein the 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, the recombinant polynucleotide 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: 48. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 48. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide encoding the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 48. In some embodiments, the recombinant polynucleotide comprises SEQ ID NO: 48. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 48 is integrated into a safe harbor locus of a cell.

[00118] iHelper#8 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) operably linked to an inducible promoter (e.g., a tetracycline inducible promoter), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide operably linked to an inducible promoter (e.g., a tetracycline inducible promoter), and (d) a polynucleotide encoding an adenovirus VA RNA gene, wherein tire 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence and matches the 5' to 3' orientation of the E4 orf6- 6/7 coding sequence. In some embodiments, an expression cassette comprising the iHelper#8 polynucleotide is integrated into a safe harbor locus of a cell. [00119] iHelper#9 In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises, in a 5' to 3' order, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) operably linked to an inducible promoter (e.g., a tetracycline inducible promoter), (b) a polynucleotide encoding an adenovirus L4 22K/33K polypeptide operably linked to an inducible promoter (e.g., a tetracycline inducible promoter), (c) a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide operably linked to an inducible promoter (e.g., a tetracycline inducible promoter), and (d) a polynucleotide encoding an adenovirus VA RNA gene, wherein the 5' to 3' orientation of the L4 22K/33K coding sequence is opposite to the 5' to 3' orientation of the E2A DBP coding sequence and matches the 5' to 3' orientation of the E4 orf6-6/7 coding sequence. In some embodiments, an expression cassette comprising the iHelper#9 polynucleotide sequence is integrated into a safe harbor locus of a cell.

[00120] iHelper#6-sRep In some embodiments, an isolated recombinant polynucleotide encoding one or more helper functions described herein comprises in a 5' to 3' order, (a) a tetracycline inducible promoter operably linked to an AAV Rcp52/40 coding region, (b) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (c) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (d) a bidirectional tetracycline inducible promoter, (e) a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide, and (f) a polynucleotide encoding an adenovirus VA RNA gene, wherein the bidirectional tetracycline inducible promoter is operably linked to the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) and the polynucleotide encoding an adenovirus E4orf6-6/7 polypeptide and wherein the 5' to 3' orientation of the gene encoding an adenovirus L4 22K/33K polypeptide is opposite to the 5' to 3' orientation of the E2A DBP coding sequence. In some embodiments, the tetracycline inducible promoter of (a) comprises the nucleotide sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the AAV Rep coding region comprises a mutation or mutations that reduce or eliminate the activity of the AAV p40 promoter. In some embodiments, the AAV Rep52/40 coding region 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: 56. In some embodiments, the AAV Rep52/40 coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 56. In some embodiments, the AAV Rep52/40 coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 56. In some embodiments, the AAV Rep52/40 coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 56. In some embodiments, the AAV Rep52/40 coding region comprises the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the recombinant polynucleotide 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: 75. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 75. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide encoding the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 75. In some embodiments, the recombinant polynucleotide comprises SEQ ID NO: 75. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 5 7is integrated into a safe harbor locus of a cell.

[00121] In some embodiments, the disclosure further provides a host cell comprising a recombinant polynucleotide described herein encoding one or more helper functions (c.g., a recombinant polynucleotide encoding an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), an inducible gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), and an adenovirus VA RNA gene). In some embodiments, the recombinant polynucleotide described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the host cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HcLa cell, SF-9 cell, BHK cell, Vcro cell, or PcrC6 cell. In some embodiments, the host cell is a EIEK293 cell or a HEK293 derived cell.

Polynucleotides encoding one or more AAV rep genes and an AAV cap gene

[00122] In some embodiments, the disclosure provides an isolated recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene. In some embodiments, the one or more AAV rep genes encode a Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, Rep40 polypeptide or any combination thereof. In some embodiments, the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, the one or more AAV rep genes and the AAV cap gene encode Rep polypeptides and capsid polypeptides that have the same serotype. In some embodiments, tire one or more AAV rep genes and the AAV cap gene encode Rep polypeptides and capsid polypeptides that have different serotypes. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV2 capsid polypeptide. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV6 capsid polypeptide. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV8 capsid polypeptide. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV9 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the AAV cap gene further encodes an assembly-activating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP).

[00123] In some embodiments, tire one or more AAV rep genes or the AAV cap gene comprises a heterologous promoter, in some embodiments, the one or more AAV rep genes comprise a heterologous promoter. In some embodiments, the AAV cap gene comprises a heterologous promoter. In some embodiments, the one or more AAV rep genes and the AAV cap gene comprise a heterologous promoter. In some embodiments, the heterologous promoter is a CMV promoter or an engineered CMV promoter. In some embodiments, the heterologous promoter is an inducible promoter. In some embodiments, the inducible promoter is an alcohol inducible promoter, a forskolin inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter, a rapamycin inducible promoter, a hormone inducible promoter and a metal ion inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter.

[00124] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises an AAV Rep coding region and an AAV cap coding region operably linked to a single promoter. In some embodiments, the recombinant polynucleotide comprises a promoter operably linked to an expression cassette comprising an AAV Rep coding region, an AAV Cap coding region and an IRES element operably linked to the AAV Rep coding region or AAV Cap coding region. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter.

[00125] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises an AAV rep gene comprising, in 5' to 3' order, (i) a first promoter, (ii) a first polynucleotide encoding the 5' portion of an AAV Rep78 and/or 68 polypeptide, (iii) an intron comprising a second promoter, and (iv) a second polynucleotide comprising overlapping coding regions encoding the 3' portion of the AAV Rep78 and/or 68 polypeptide and an AAV Rep52 and/or Rep40 polypeptide, wherein the first promoter is operably linked to the first polynucleotide and the second promoter is operably linked to the second nucleotide. In some embodiments, the first and second polynucleotide comprises a mutation or mutations that reduce or eliminate tire activity of the AAV p 19 and p40 promoters. In some embodiments, the first and second polynucleotide comprises a mutation or mutations that reduce or eliminate the activity of the AAV p!9 and p40 promoters without altering the amino acid sequence of the Rep polypeptides. In some embodiments, the first and/or second promoter is an inducible promoter. In some embodiments, the first and second promoters are inducible promoters. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the AAV rep gene encodes (i) a Rep78 and Rep52 polypeptides; (ii) a Rep68 and Rep40 polypeptides, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptides. In some embodiments, tire AAV rep gene encodes a Rcp78, Rcp68, Rcp52 and Rcp40 polypeptides. AAV Rep polypeptides

[00126] In some embodiments, an isolated recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene comprises an AAV Rep gene that has been modified relative to the naturally occurring AAV2 Rep gene. In some embodiments, the AAV Rep gene that has been modified by replacing the p5 promoter with a heterologous promoter, replacing the promoter with an IRES element, truncating the rep polypeptide coding region, introducing a mutation or mutations that reduce or eliminate the activity of tire AAV p!9 and/or p40 promoters (e.g., without altering the amino acid sequence of the Rep polypeptides), inserting an intron comprising a promoter (e.g., an inducible promoter) wherein the promoter in the intron is operably linked to the Rep52/40 coding region, and any combinations thereof. Figure 11 . A skilled artisan understands that these modifications can be used to create an AAV Rep gene that expresses one or more of the Rcp78, Rcp68, Rcp52 and Rcp40 polypeptides. In some embodiments, an AAV Rep gene described herein expresses one Rep polypeptide (e.g., Rep78, Rep68, Rep52 or Rep40). In some embodiments, an AAV Rep gene described herein expresses two Rep polypeptides (e.g., Rep 8 and Rep68, Rep78 and Rep52, Rep68 and Rep40, and Rep52 and Rep40). In some embodiments, an AAV Rep gene described herein expresses all four Rep polypeptides (i.e., Rep78, Rep68, Rep52 and Rep40).

[00127] In some embodiments, an AAV Rep gene described herein comprises one or more inducible promoters. In some embodiments, an AAV Rep gene described herein comprises an inducible promoter at the 5' end of tire AAV Rep polypeptide coding region. In some embodiments, an AAV Rep gene described herein comprises an intron in the AAV Rep polypeptide coding region, wherein the intron comprises an inducible promoter. In some embodiments, an AAV Rep gene described herein comprises (i) an inducible promoter at the 5' end of the AAV Rep polypeptide coding region and (ii) an intron comprising an inducible promoter. In some embodiments, the inducible promoter is an alcohol inducible promoter, a forskolin inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter, a rapamycin inducible promoter, a hormone inducible promoter and a metal ion inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible 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 to SEQ ID NO: 1, 2 or 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises tire nucleotide sequence of SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, tire tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 3. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3.

[00128] In some embodiments, an AAV Rep gene described herein comprises one or more CMV promoter or an engineered CMV promoter or transcriptionally active fragment thereof. In some embodiments, the promoter is a CMV promoter. In some embodiments, the CMV 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 to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

Engineered CMV immediate early promoters or transcriptionally active fragments thereof are known to one of skill, for example, as disclosed in International Publication No.

WO2023141582A1, which is incorporated herein by reference in its entirety.

[00129] An AAV rep gene described herein can encode a Rep78 polypeptide of any serotype. In some embodiments, an AAV Rep gene described herein encodes one or more AAV2 rep polypeptides.

[00130] In some embodiments, an AAV2 Rep78 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 to SEQ ID NO: 49. In some embodiments, the AAV2 Rep78 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 49. In some embodiments, the AAV2 Rep78 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 49. In some embodiments, the AAV2 Rep78 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 49. In some embodiments, the AAV2 Rep78 polypeptide comprises the amino acid sequence of SEQ ID NO: 49.

[00131] In some embodiments, an AAV2 Rep68 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 to SEQ ID NO: 50. In some embodiments, the AAV2 Rep68 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 50. In some embodiments, the AAV2 Rep68 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 50. In some embodiments, the AAV2 Rep68 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 50. In some embodiments, the AAV2 Rep68 polypeptide comprises the amino acid sequence of SEQ ID NO: 50.

[00132] In some embodiments, an AAV2 Rep52 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 to SEQ ID NO: 51. In some embodiments, the AAV2 Rep52 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 51. In some embodiments, the AAV2 Rep52 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 51. In some embodiments, the AAV2 Rep52 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 51. In some embodiments, the AAV2 Rep52 polypeptide comprises the amino acid sequence of SEQ ID NO: 51.

[00133] In some embodiments, an AAV2 Rep40 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 to SEQ ID NO: 52. In some embodiments, the AAV2 Rcp40 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 52. In some embodiments, the AAV2 Rep40 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 52. In some embodiments, the AAV2 Rep40 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 52. In some embodiments, the AAV2 Rep40 polypeptide comprises the amino acid sequence of SEQ ID NO: 52.

[00134] pl9 and/or p40 mutations In some embodiments, an AAV Rep gene described herein comprises a wild type AAV Rep coding region that encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide. In some embodiments, an AAV Rep gene described herein comprises a wild type AAV2 Rep coding region 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: 53. In some embodiments, tire wild type AAV2 Rep coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 53. In some embodiments, the wild type AAV2 Rep coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 53. In some embodiments, the wild type AAV2 Rep coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 53. In some embodiments, the wild type AAV2 Rep coding region comprises the nucleotide sequence of SEQ ID NO: 53. [00135] In some embodiments, an AAV Rep gene described herein comprises an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 or AAV pl9 and/or p40 promoters. In some embodiments, an AAV Rep gene described herein comprises an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 or AAV pl9 and/or p40 promoters without altering the amino acid sequence of the Rep polypeptides. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 promoter comprises the A1506C and T1509C substitutions, wherein the residue numbering is according to SEQ ID NO: 53, i.e., the A to T and T to C substitutions are at the nucleotide position corresponding to residue 1506 and 1509, respectively, of SEQ ID NO: 53. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of tire AAV p40 promoter comprises the T1503C, A1506C. T1509C and G1512A substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV pl9 promoter comprises the T510C, T513C, T525C, T526C, and A528C substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of tire AAV pl9 and/or p40 promoters comprises the T510C, T513C, T525C, T526C, A528C, T1503C, A1506C, T1509C and G1512A substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 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 to SEQ ID NO: 54, optionally wherein the AAV Rep coding region encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 54. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 54. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 54. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 promoter comprises the nucleotide sequence of SEQ ID NO: 54. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p!9 and/or p40 promoters 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: 55, optionally wherein the AAV Rep coding region encodes a Rep78 and Rep68 polypeptide. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate tire activity of the AAV p!9 and/or p40 promoters comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 55. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV pl9 and/or p40 promoters comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 55. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate tire activity of the AAV p!9 and/or p40 promoters comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 55. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p!9 and/or p40 promoters comprises the nucleotide sequence of SEQ ID NO: 55. In some embodiments, an AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p40 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 to SEQ ID NO: 56, optionally wherein the AAV Rep coding region encodes a Rep52 and Rep40 polypeptide. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p!9 and/or p40 promoters comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 56. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV pl9 and/or p40 promoters comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 56. In some embodiments, the AAV Rep coding region comprising a mutation or mutations that reduce or eliminate the activity of the AAV p!9 and/or p40 promoters comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 56. In some embodiments, the AAV Rep coding region comprising a mutation or

Ill mutations that reduce or eliminate the activity of the AAV pl 9 and/or p40 promoters comprises the nucleotide sequence of SEQ ID NO: 56.

[00136] Intron comprising a promoter In some embodiments, an AAV Rep gene described herein comprises an AAV Rep polypeptide coding region encoding one or more large rep polypeptides (e.g.. Rep78 and/or Rep68) and one or more small rep polypeptides (e.g., Rep52 and/or Rep40), wherein the coding region comprises an intron upstream from the region encoding the one or more small rep polypeptides, wherein the intron comprises a promoter (e.g., tetracycline inducible promoter) operably linked to the region encoding the one or more small rep polypeptide. A skilled artisan understand that the coding region encoding the one or more small rep polypeptides also encodes the C terminal portion of the one or more large rep polypeptides. In some embodiments, an AAV Rep gene described herein comprises an AAV Rep polypeptide coding region encoding a Rep78 and Rep52 polypeptide, wherein the coding region comprises an intron upstream from the region encoding the Rep52 polypeptide, wherein the intron comprises a promoter (e.g., tetracycline inducible promoter) operably linked to the region encoding the Rep52 polypeptide. In some embodiments, an AAV Rep gene described herein comprises an AAV Rep polypeptide coding region encoding a Rep68 and Rep40 polypeptide, wherein the coding region comprises an intron upstream from the region encoding the Rep40 polypeptide, wherein the intron comprises a promoter (e.g., tetracycline inducible promoter) operably linked to the region encoding the Rep40 polypeptide. In some embodiments, the promoter is a tetracycline inducible promoter. In some embodiments, the intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a branching site and acceptor site comprising a nucleotide sequence of SEQ ID NO: 109 or 103 comprising 0, 1, 2, 3, 4 or 5 substitutions. In some embodiments, the intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a branching site and acceptor site comprising the nucleotide sequence of SEQ ID NO: 109 or 103. In some embodiments, the intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a 3' arm comprising a branching site and acceptor site, wherein the 3' arm 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, 112 or 115. In some embodiments, the intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a 3' arm comprising a branching site and acceptor site, wherein the 3' arm comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 110, 112 or 115. In some embodiments, the intron comprises ( 1 ) a splicing donor site of GTAAGT or GTAAGA and (2) a 3' arm comprising a branching site and acceptor site, wherein the 3' arm comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 110, 112 or 115. In some embodiments, tire intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a 3' arm comprising a branching site and acceptor site, wherein the 3' arm comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 110. 112 or 115. In some embodiments, the intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a 3' arm comprising a branching site and acceptor site, wherein the 3' arm comprises the nucleotide sequence of SEQ ID NO: 110, 112 or 115. In some embodiments, tire intron comprises (1) a splicing donor site of GTAAGT or GTAAGA and (2) a 3' ann comprising a branching site and acceptor site, wherein the 3' arm 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 intron comprises (1) 5' arm comprising a splicing donor site and (2) a 3' arm comprising a branching site and acceptor site, wherein the 5' arm 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 or 114 and the 3' ann 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, 112 or 115. In some embodiments, the 5' arm comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 111 or 114 and the 3' arm comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 110, 112 or 115. In some embodiments, the 5' arm comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 111 or 114 and the 3' ann comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 110, 112 or 115. In some embodiments, the 5' arm comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 111 or 114 and the 3' arm comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 110, 112 or 115. In some embodiments, the 5' arm comprises the nucleotide sequence of SEQ ID NO: 111 or 114 and the 3' arm comprises the nucleotide sequence of SEQ ID NO: 110, 112 or 115. In some embodiments, the 5' ann 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 and the 3' arm 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, 112. the 5' arm 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 and the 3' arm 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 intron comprising an inducible 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 to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises the nucleotide sequence of SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible 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 to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 57, 58, 59, or 60 is integrated into a safe harbor locus of a cell. In some embodiments, the intron reduces or eliminates the activity of the pl9 promoter. In some embodiments, the intron is inserted between the TATA box and the TATA-20 element of the p!9 promoter and reduces or eliminates the activity of the p 19 promoter. In some embodiments, the intron is inserted at the nucleotide position corresponding to residue 530 of SEQ ID NO: 53 and reduces or eliminates the activity of the p!9 promoter. In some embodiments, the AAV Rep coding region comprises a mutation or mutations that reduce or eliminate the activity of the p40 promoter. In some embodiments, the mutation or mutations that reduce or eliminate the activity of the p40 promoter comprise the A1506C and T1509C substitutions, orthe T1503C, A1506C, T1509C and G1512A substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, the AAV Rep polypeptide coding region 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: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 61, 62. 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises the nucleotide sequence of SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region 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: 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 64. In some embodiments, the AAV Rep polypeptide coding region comprises the nucleotide sequence of SEQ ID NO: 64. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 61, 62, 63, or 64 is integrated into a safe harbor locus of a cell.

AAV capsid polypeptides

[00137] In some embodiments, the disclosure provides an isolated recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene, wherein the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, the AAV cap gene further encodes an assembly-activating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP).

[00138] In some embodiments, the AAV cap gene comprises a heterologous promoter. In some embodiments, the heterologous promoter is an inducible promoter. In some embodiments, the inducible promoter is an alcohol inducible promoter, a forskolin inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter, a rapamycin inducible promoter, a hormone inducible promoter and a metal ion inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible 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 to SEQ ID NO: 1, 2 or 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 1 . In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises tire nucleotide sequence of SEQ ID NO: 2. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:

[00139] In some embodiments, the AAV cap gene comprises a heterologous promoter, wherein the heterologous promoter is a CMV promoter or an engineered CMV promoter or transcriptionally active fragment thereof. In some embodiments, the promoter is a CMV promoter. In some embodiments, the CMV 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 to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 4. In some embodiments, the CMV promoter comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the promoter is an engineered CMV promoter or transcriptionally active fragment thereof. Engineered CMV immediate early promoters or transcriptionally active fragments thereof are known to one of skill, for example, as disclosed in International Publication No. WO2023141582A1. which is incorporated herein by reference in its entirety.

[00140] An isolated recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene can comprise an AAV cap gene encoding a VP1, VP2 and VP3 capsid polypeptide of any serotype. In some embodiments, the AAV cap gene comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. AAV11, AAV12, AAV13. AAV14, AAV15 and AAV16, AAV.rh8. AAV.rh lO, 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 AAV cap gene comprises a serotype selected from the group consisting of AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. In some embodiments, the AAV cap gene comprises a serotype selected from the group consisting of AAV8 or AAV9 serotype. In some embodiments, an expression cassette comprising a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene is integrated into a safe harbor locus of a cell.

[00141] In some embodiments, the AAV cap gene comprises the AAV2 serotype. In some embodiments, 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 to SEQ ID NO: 65. In some embodiments, the AAV2 cap gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 65. In some embodiments, the AAV2 cap gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 65. In some embodiments, the AAV2 cap gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 65. In some embodiments, the AAV2 cap gene comprises the nucleotide sequence of SEQ ID NO: 65. In some embodiments, an expression cassette comprising the AAV2 cap gene is integrated into a safe harbor locus of a cell.

[00142] In some embodiments, the AAV cap gene comprises the AAV6 serotype. In some embodiments, 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 to SEQ ID NO: 66. In some embodiments, the AAV6 cap gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 66. In some embodiments, the AAV6 cap gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 66. In some embodiments, the AAV6 cap gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 66. In some embodiments, the AAV6 cap gene comprises the nucleotide sequence of SEQ ID NO:

66. In some embodiments, an expression cassette comprising the AAV6 cap gene is integrated into a safe harbor locus of a cell.

[00143] In some embodiments, the AAV cap gene comprises the AAV8 serotype. In some embodiments, 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 to SEQ ID NO: 67. In some embodiments, the AAV8 cap gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 67. In some embodiments, the AAV8 cap gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 67. In some embodiments, the AAV8 cap gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 67. In some embodiments, the AAV8 cap gene comprises the nucleotide sequence of SEQ ID NO:

67. In some embodiments, an expression cassette comprising the AAV8 cap gene is integrated into a safe harbor locus of a cell.

[00144] In some embodiments, the AAV cap gene comprises the AAV9 serotype. In some embodiments, 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 to SEQ ID NO: 68. In some embodiments, the AAV9 cap gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 68. In some embodiments, the AAV9 cap gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 68. In some embodiments, the AAV9 cap gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 68. In some embodiments, the AAV9 cap gene comprises the nucleotide sequence of SEQ ID NO: 68. In some embodiments, an expression cassette comprising the AAV9 cap gene is integrated into a safe harbor locus of a cell.

Trans polynucleotides

[00145] In some embodiments, the disclosure provides an isolated recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene. In some embodiments, the one or more AAV rep genes and the AAV cap gene encode Rep polypeptides and capsid polypeptides that have the same serotype. In some embodiments, the one or more AAV rep genes and the AAV cap gene encode Rep polypeptides and capsid polypeptides that have different serotypes. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV2 capsid polypeptide. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV6 capsid polypeptide. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV8 capsid polypeptide. In some embodiments, the one or more AAV rep genes encode AAV2 Rep polypeptides and the AAV cap gene encodes AAV9 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene.

[00146] In some embodiments, the one or more AAV rep genes or the AAV cap gene comprises a heterologous promoter. In some embodiments, the one or more AAV rep genes comprise a heterologous promoter. In some embodiments, the AAV cap gene comprises a heterologous promoter. In some embodiments, the one or more AAV rep genes and the AAV cap gene comprise a heterologous promoter. In some embodiments, the heterologous promoter is a CMV promoter or an engineered CMV promoter. In some embodiments, the heterologous promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter.

[00147] iTrans#5 In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises an AAV Rep coding region and an AAV Cap coding region operably linked to a single promoter. In some embodiments, the recombinant polynucleotide comprises a promoter operably linked to an expression cassette comprising an AAV Rep coding region, an AAV Cap coding region and an IRES element operably linked to the AAV Rep coding region or AAV Cap coding region. In some embodiments, the expression cassette comprises, in 5' to 3' order, the AAV Cap coding region, the internal ribosome entry site (IRES), and the AAV Rep coding region. In some embodiments, the AAV Cap coding region encodes a VP1, VP2 and VP3 polypeptide. In some embodiments, the AAV Cap coding region comprises a serotype selected from the group consisting of AAV2, AAV6, AAV8 and AAV9. In some embodiments, the AAV Rep coding region encodes one or more of a Rep78, Rep68, Rep52 and Rep40 polypeptide. In some embodiments, the AAV Rep coding region comprises a mutation or mutations that reduces or eliminates the activity of the AAV p40 promoter. In some embodiments, the AAV Rep coding region comprises mutations that reduces or eliminates the activity of the AAV pl9 and p40 promoters. In some embodiments, AAV Rep coding region comprises an ACG start codon in place of ATG. In some embodiments, the AAV Rep coding region comprises an AU rich mRNA destabilizing (AREs) in the 3' untranslated region. Pharm et al., BioTechniques 45(2): 155-162 (2008). In some embodiments, the AAV Rep coding region comprises a scrambled Rep coding region that was created by shuffling synonymous codon pairs while maintaining codon use and the free energy of folded RNA to prevent large changes in secondary structure. Sitaraman ct al., PNAS 108(34): 14294- 14299 (2011). In some embodiments, the expression cassette further comprises a poly A signal. [00148] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible 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 to SEQ ID NO: 1, 2 or 3. In some embodiments, the tetracycline inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 1, 2 or 3. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1, 2 or 3.

[00149] A skilled artisan understand that any IRES capable of promoting translation can be used in the context of a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein. In some embodiments, the IRES 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: 69. In some embodiments, the IRES comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 69. In some embodiments, the IRES comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 69. In some embodiments, the IRES comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 69. In some embodiments, the IRES comprises the nucleotide sequence of SEQ ID NO: 69. [00150] In some embodiments, the AAV Rep coding region encodes (i) a Rep 78 and Rep 52 polypeptide, (ii) Rep 68 and Rep 40 polypeptide, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptide, wherein the AAV Rep coding region comprises a functional pl9 promoter. In some embodiments, the AAV Rep coding region encodes a Rep 78 and Rep 52 polypeptide. In some embodiments, the AAV Rep coding region encodes a Rep 68 and Rep 40 polypeptide. In some embodiments, the AAV Rep coding region encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide.

[00151] In some embodiments, the AAV Rep coding region encodes (i) a Rep78 polypeptide, (ii) Rep 68 polypeptide, or (iii) a Rep78 and Rep 68 polypeptide, wherein the AAV Rep coding region comprises a mutation or mutations that reduce or eliminate p 19 promoter activity. In some embodiments, the AAV Rep coding region encodes a Rep78 polypeptide. In some embodiments, the AAV Rep coding region encodes a Rep68 polypeptide. In some embodiments, the AAV Rep coding region encodes a Rcp78 and Rcp68 polypeptide.

[00152] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises an inducible promoter (e g., tetracycline inducible promoter) operably linked to an expression cassette, wherein the expression cassette comprises, in 5' to 3' order, an AAV Cap coding region, an internal ribosome entry site (IRES), and an AAV Rep coding region, wherein the AAV Cap coding region encodes a VP1, VP2 and VP3 polypeptide, and wherein the AAV Rep coding region (i) encodes a Rcp78, Rcp68, Rcp52 and Rep40 polypeptide, and (ii) comprises one or more mutations that reduce or eliminate p40 promoter activity. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the AAV Cap coding region comprises a serotype selected from the group consisting of AAV2, AAV6, AAV8 and AAV9. In some embodiments, the AAV Rep coding region 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: 54. In some embodiments, the AAV Rep coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 54. In some embodiments, the AAV Rep coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 54. In some embodiments, the AAV Rep coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 54. In some embodiments, the AAV Rep coding region comprises the nucleotide sequence of SEQ ID NO: 54. In some embodiments, the expression cassette 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: 70. In some embodiments, the expression cassette comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 70. In some embodiments, the expression cassette comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 70. In some embodiments, the expression cassette comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 70. In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO: 70. In some embodiments, the recombinant polynucleotide 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: 71 . In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 71. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 71. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 71. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 71. In some embodiments, the expression cassette further comprises a poly A signal. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 71 is integrated into a safe harbor locus of a cell.

[00153] iTrans#10 In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises an inducible promoter (e.g., tetracycline inducible promoter) operably linked to an expression cassette, wherein the expression cassette comprises, in 5' to 3' order, an AAV Cap coding region, an internal ribosome entry site (IRES), and an AAV Rep coding region, wherein the AAV Cap coding region encodes a VP1, VP2 and VP3 polypeptide, and wherein the AAV Rep coding region (i) encodes a Rep78 and Rep68 polypeptide, and (ii) comprises one or more mutations that reduce or eliminate the p 19 and p40 promoter activity. In some embodiments, the AAV Cap coding region further comprises an intron immediately upstream from the VP 1 start codon. In some embodiments, the intron 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: 74. In some embodiments, the intron comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 74. In some embodiments, the intron comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 74. In some embodiments, the intron comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 74. In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO: 74. In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the AAV Cap coding region comprises a serotype selected from the group consisting of AAV2, AAV6, AAV8 and AAV9. In some embodiments, the AAV Rep coding region 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: 55. In some embodiments, the AAV Rep coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 55. In some embodiments, the AAV Rep coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 55. In some embodiments, the AAV Rep coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 55. In some embodiments, the AAV Rep coding region comprises the nucleotide sequence of SEQ ID NO: 55. In some embodiments, the expression cassette 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: 72. In some embodiments, the expression cassette comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 72. In some embodiments, tire expression cassette comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 72. In some embodiments, the expression cassette comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 72. In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO: 72. In some embodiments, the recombinant polynucleotide 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: 73. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 73. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 73. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 73. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 73. In some embodiments, the expression cassette further comprises a poly A signal. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 73 is integrated into a safe harbor locus of a cell.

[00154] iTrans#6/7/8/7TetCap In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises (I) an AAV rep gene comprising, in 5' to 3' order, (i) a first promoter, (ii) a first polynucleotide encoding the 5' portion of an AAV Rep78 and/or 68 polypeptide, (iii) an intron comprising a second promoter, and (iv) a second polynucleotide comprising overlapping coding regions encoding the 3' portion of the AAV Rep78 and/or 68 polypeptide and an AAV Rep52 and/or Rep40 polypeptide, wherein the first promoter is operably linked to the first polynucleotide and the second promoter is operably linked to the second nucleotide, and (2) a third promoter operably linked to an AAV Cap coding region, wherein the AAV Cap coding region encodes a VP1, VP2 and VP3 polypeptide. In some embodiments, the intron reduces or eliminates the activity of the p!9 promoter. In some embodiments, the intron is inserted between the TATA box and the TATA-20 element of the p!9 promoter and reduces or eliminates the activity of the p 19 promoter. In some embodiments, the intron is inserted at the nucleotide position corresponding to residue 530 of SEQ ID NO: 53 and reduces or eliminates the activity of the p 19 promoter. In some embodiments, the first and second polynucleotide comprises a mutation or mutations that reduce or eliminate the activity of the AAV p!9 and/or p40 promoters. In some embodiments, the first and second polynucleotide comprises a mutation or mutations that reduce or eliminate the activity of the AAV pl9 and/or p40 promoters without altering the amino acid sequence of tire Rep polypeptides. In some embodiments, the mutation or mutations that reduce or eliminate the activity of the p40 promoter comprise the A1506C and T1509C substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, the mutation or mutations that reduce or eliminate the activity of the p40 promoter comprises the T1503C, A1506C, T1509C and G1512A substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, the mutation or mutations that reduce or eliminate the activity of the p 19 promoter comprises the T510C, T513C. T525C. T526C, and A528C substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, the mutation or mutations that reduce or eliminate the activity of the AAV pl9 and/or p40 promoters comprises the T510C, T513C, T525C, T526C, A528C, T1503C, A1506C, T1509C and G1512A substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, tire AAV rep gene encodes (i) a Rep78 and Rep52 polypeptides; (ii) a Rep68 and Rep40 polypeptides, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptides. In some embodiments, the AAV rep gene encodes Rep78 and Rep52 polypeptides. In some embodiments, the AAV rep gene encodes a Rep78, Rep68. Rep52 and Rep40 polypeptides. In some embodiments, the intron comprising an inducible 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 to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises the nucleotide sequence of SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the first polynucleotide 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: 61, 62, 63 or 64. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the first and/or second promoter is an inducible promoter (e.g., a tetracycline inducible promoter). In some embodiments, the first and second promoters are inducible promoters (e.g., tetracycline inducible promoters). In some embodiments, tire inducible promoter is a tetracycline inducible promoter. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the third promoter is a CMV promoter. In some embodiments, the promoter is an engineered CMV promoter or transcriptionally active fragment thereof. In some embodiments, the CMV 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 to SEQ ID NO: 76. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 76. In some embodiments, tire CMV promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 76. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 76. In some embodiments, the CMV promoter comprises the nucleotide sequence of SEQ ID NO: 76. In some embodiments, the third promoter is an inducible promoter (e.g., a tetracycline inducible promoter). In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the AAV Cap coding region comprises a serotype selected from the group consisting of AAV2, AAV6, AAV8 and AAV9. In some embodiments, the recombinant polynucleotide encoding one or more AAV rep genes and an AAV 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 to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 77, 78, 79. 80. 81 or 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 77, 78. 79. 80, 81 or 82 is integrated into a safe harbor locus of a cell.

[00155] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises, in 5' to 3' order, an AAV Rep gene described herein comprising (i) a first promoter operably linked to (ii) an AAV Rep polypeptide coding region encoding one or more large rep polypeptides (e.g., Rep78 and/or Rep68) and one or more small rep polypeptides (e.g.. Rep52 and/or Rep40), wherein the coding region comprises an intron upstream from the region encoding the one or more small rep polypeptides, wherein the intron comprises a second promoter (e.g., tetracycline inducible promoter) operably linked to the region encoding the one or more small rep polypeptide and an AAV Cap gene comprising (iii) a third promoter operably linked to (iv) an AAV Cap coding region, wherein the AAV Cap coding region encodes a VP1, VP2 and VP3 polypeptide. In some embodiments, the AAV Rep polypeptide coding region encoding a Rep78 and Rep52 polypeptide comprises an intron upstream from the region encoding the Rep52 polypeptide, wherein the intron comprises a second promoter (e.g., tetracycline inducible promoter) operably linked to the region encoding the Rep52 polypeptide. In some embodiments, the AAV Rep polypeptide coding region encoding a Rep68 and Rep40 polypeptide comprises an intron upstream from the region encoding the Rep40 polypeptide, wherein the intron comprises a second promoter (e.g., tetracycline inducible promoter) operably linked to the region encoding the Rep40 polypeptide. In some embodiments, the promoter is a tetracycline inducible promoter. In some embodiments, the intron comprising an inducible 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 to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible promoter comprises the nucleotide sequence of SEQ ID NO: 57, 58, 59 or 60. In some embodiments, the intron comprising an inducible 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 to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 60. In some embodiments, tire intron comprising an inducible promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 60. In some embodiments, the intron comprising an inducible promoter comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the intron reduces or eliminates the activity of the p 19 promoter. In some embodiments, the intron is inserted between the TATA box and the TATA-20 element of the p!9 promoter and reduces or eliminates the activity of the p 19 promoter. In some embodiments, the intron is inserted at the nucleotide position corresponding to residue 530 of SEQ ID NO: 53 and reduces or eliminates the activity of the pl9 promoter. In some embodiments, the AAV Rep coding region comprises a mutation or mutations that reduce or eliminate the activity of the p40 promoter. In some embodiments, the mutation or mutations that reduce or eliminate the activity of the p40 promoter comprise tire A1506C and T1509C substitutions, orthe T1503C, A1506C, T1509C and G1512A substitutions, wherein the residue numbering is according to SEQ ID NO: 53. In some embodiments, the AAV Rep polypeptide coding region 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: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 61 , 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region comprises the nucleotide sequence of SEQ ID NO: 61, 62, 63 or 64. In some embodiments, the AAV Rep polypeptide coding region 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: 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 64. In some embodiments, the AAV Rep polypeptide coding region comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 64. In some embodiments, the AAV Rep polypeptide coding region comprises the nucleotide sequence of SEQ ID NO: 64. In some embodiments, the first and/or second promoter is an inducible promoter (e.g., a tetracycline inducible promoter). In some embodiments, the first and second promoters are inducible promoters (e.g., tetracycline inducible promoters). In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the third promoter is a CMV promoter. In some embodiments, the promoter is an engineered CMV promoter or transcriptionally active fragment thereof. In some embodiments, the CMV 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 to SEQ ID NO: 76. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 76. In some embodiments, tire CMV promoter comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 76. In some embodiments, the CMV promoter comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 76. In some embodiments, the CMV promoter comprises the nucleotide sequence of SEQ ID NO: 76. In some embodiments, the third promoter is an inducible promoter (e.g., a tetracycline inducible promoter). In some embodiments, the tetracycline inducible promoter comprises the nucleotide sequence of SEQ ID NO: 1, 2 or 3. In some embodiments, the AAV Cap coding region comprises a serotype selected from the group consisting of AAV2, AAV6, AAV8 and AAV9. In some embodiments, the recombinant polynucleotide encoding one or more AAV rep genes and an AAV 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 to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 77, 78, 79, 80, 81 or 82. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00156] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene 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 to SEQ ID NO: 77. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 77. In some embodiments, tire recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 77. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 77. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 77. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00157] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene 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 to SEQ ID NO: 78. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 78. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 78. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 78. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 78. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00158] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene 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 to SEQ ID NO: 79. In some embodiments, tire recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 79. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 79. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 79. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 79. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00159] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene 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 to SEQ ID NO: 80. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 80. In some embodiments, tire recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 80. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 80. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00160] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene 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 to SEQ ID NO: 81. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 81. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 81. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 81. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 81. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00161] In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene 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 to SEQ ID NO: 82. In some embodiments, tire recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 82. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 82. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 82. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00162] 3.4 to 5.4 In some embodiments, a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein comprises, in 5' to 3' order, a first promoter operably linked to a first AAV Rep coding region and a second promoter operably linked to an AAV Cap coding region, wherein (i) the first AAV rep coding region comprises a functional pl9 promoter and encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide, (ii) the first AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity: and (iii) the AAV cap coding region comprises an AAV Cap intron and encodes a VP1, VP2 and VP3 polypeptide. In some embodiments, the recombinant polynucleotide comprises in 5' to 3' order a first promoter operably linked to a first AAV Rep coding region, a second promoter operably linked to an AAV Cap coding region and a third promoter operably linked to a second AAV Rep coding region, wherein the second AAV Rep coding region encodes a Rep52 and Rep40 polypeptide, and wherein second AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity. In some embodiments, one or more of the first AAV Rep coding region, second AAV Rep coding region and AAV Cap coding region comprises a poly A signal. In some embodiments, one or more of the first, second and third is an inducible promoter. In some embodiments, the inducible promoter is an alcohol inducible promoter, a forskolin inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter, a rapamycin inducible promoter, a hormone inducible promoter and a metal ion inducible promoter. In some embodiments, tire inducible promoter is a tetracycline inducible promoter. In some embodiments, the tetracycline inducible 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 to SEQ ID NO: 1 , 2 or 3. In some embodiments, the recombinant polynucleotide encoding one or more AAV rep genes and an AAV 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 to SEQ ID NO: 83-90 or 91. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 83-90 or 91. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 83- 90 or 91. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 83-90 or 91. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 83-90 or 91. In some embodiments, the AAV Rep coding region and the AAV Cap coding region comprise a poly A signal.

[00163] In some embodiments, the disclosure further provides a host cell comprising a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene described herein. In some embodiments, the recombinant polynucleotide described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the host cell is a HEK293 cell, HEK derived cell. CHO cell, CHO derived cell. HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the host cell is a HEK293 cell or a HEK293 derived cell. Packaging polynucleotides encoding one or more AAV rep genes, an AAV cap gene, and one or more helper functions

[00164] [0049] [0053] In some embodiments, the disclosure provides isolated packaging polynucleotides encoding one or more helper functions, one or more AAV rep genes, and an AAV cap gene. In some embodiments, a packaging polynucleotide described herein is capable of supporting the production of recombinant AAV particles in a host cell (e.g., HEK293 cell) comprising a cis vector or polynucleotide encoding the rAAV genome . In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), and an adenovirus VA RNA gene. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the one or more AAV rep genes encode a Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, Rep40 polypeptide or any combination thereof. In some embodiments, the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the AAV cap gene further encodes an assemblyactivating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP).

[00165] In some embodiments, the packaging polynucleotide comprises one or more recognition sites for an integrase or recombinase such that the packaging polynucleotide can be integrated into a genomic landing site of a host cell (e.g., EIEK293 cell) comprising the complementary' recognition site. In some embodiments, the packaging polynucleotide comprises two recognition sites flanking the sequences encoding one or more helper functions, one or more AAV rep genes, and an AAV cap gene. Any recognition site and integrase/recombinase known to a skilled artisan can be used. In some embodiments, the recognition site is an aft site and tire integrase is PhiC31. In some embodiments, the recognition site is an LHA and/or RHA site and the integrase is Bxbl. In some embodiments, the recognition site is a loxP site and the recombinase is Cre. In some embodiments, the recognition site is an FRT site and tire recombinase is FLP. [00166] In some embodiments, packaging polynucleotide comprises in a 5 ’to 3’ direction, (1) a tetracycline inducible promoter operably linked to (2) a Rep cassette comprising, from 5’ to 3’, an AAV Rep78/68 coding region and a Rep52/40 coding region , and (3) a CMV promoter operably linked to (4) an AAV Cap coding region, operably linked to (5) a Helper cassette comprising, from 5’ to 3’, (a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP), (b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) an inducible promoter (e.g., tetracycline inducible promoter) operably linked to a polynucleotide encoding an adenovirus E4 polypeptide (e.g., E4 orf6-6/7 or E4 orf6 polypeptide), and (d) a polynucleotide encoding an adenovirus VA RNA gene, wherein the 5' to 3' orientation of both the polynucleotide encoding the E2A DBP and the polynucleotide encoding the VA RNA are opposite to tire 5' to 3' orientation of the adenovirus L4 22K/33K polynucleotide and the E4 orf polynucleotide. In some embodiments, the AAV Rep78/68 coding region and Rep52/40 coding region is an AAV2 Rep78/68 coding region and Rep52/40 coding region. In some embodiments, the Rep 78/68 and Rep52/40 coding region comprises an inducible Rep 78/68 and Rcp52/40 coding region disclosed herein. In some embodiments, the Rep 78/68 and Rcp52/40 coding region comprises the Rep 78/68 and Rep52/40 coding region of iTrans#5, iTrans#10, iTrans#6/7/8/7TetCap. In some embodiments, the Rep 78/68 and Rep52/40 coding region comprises the Rep 78/68 and Rep52/40 coding region of 3.4-3.7 or 5.0-5.4. In some embodiments, the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises an E2A promoter operably linked to an E2A coding region. In some embodiments, the polynucleotide encoding an adenovirus E2A DNA binding protein (DBP) comprises an inducible promoter (e.g., tetracycline inducible promoter) operably linked to an E2A coding region. In some embodiments, the polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide comprises a L4 22K/33K promoter operably linked to a L4 22K/33K coding region. In some embodiments, the polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide comprises an inducible promoter (e.g., tetracycline inducible promoter) operably linked to a L4 22K/33K coding region. In some embodiments, tire packaging polynucleotide comprises flanking recognition sites for an integrase or recombinase or homology arms such that the packaging polynucleotide can be integrated into a genomic locus landing site of a host cell (e.g., HEK293 cell) comprising the complementary recognition site. [00167] In some embodiments, the packaging polynucleotide comprises a 5' fragment encoding the one or more helper functions and a 3' fragment encoding the one or more AAV rep genes and AAV cap gene. In some embodiments, the packaging polynucleotide comprises a 5' fragment encoding the one or more AAV rep genes and AAV cap gene and a 3' fragment encoding the one or more helper functions.

[00168] In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Original iHelper or iHelper# 1-9 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Original iHelper, Helper# 1 -7 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Original iHelper described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#! described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#2 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Hclpcr#3 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#4 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#5 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#6 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#7 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#8 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding Helper#9 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises any one of SEQ ID NO: 48 and 93-99. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 48. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 93. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 94. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 95. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein tire polynucleotide comprises SEQ ID NO: 96. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 97. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 98. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more helper functions, wherein the polynucleotide comprises SEQ ID NO: 99. In some embodiments, the packaging polynucleotide comprises flanking recognition sites for an integrase or recombinase such that tire packaging polynucleotide can be integrated into a genomic landing site of a host cell (e.g., HEK293 cell) comprising the complementary recognition site. [00169] In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#5 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#10 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#6 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#7 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#8 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 70. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 71. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 72. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 73. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 77. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 78. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 79. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 80. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 81. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises SEQ ID NO: 82. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding 3.4- 3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises flanking homology arms or recognition sites for an integrase or recombinase such that the packaging polynucleotide can be integrated into a genomic locus or landing site of a host cell (e.g., HEK293 cell) comprising the complementary recognition site. In some embodiments, an expression cassette comprising a nucleotide sequence having at least 98 % identity to SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90 or 91 is integrated into a safe harbor locus of a cell.

[00170] In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding Original iHelper or iHelper# 1-9 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding Original iHelper or iHelper# 1-9 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#6 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#6 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHclpcr#8 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein tire first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises SEQ ID NO: 48 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises SEQ ID NO: 48 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91 . In some embodiments, the packaging polynucleotide comprises flanking homology arms or recognition sites for an integrase or recombinase such that the packaging polynucleotide can be integrated into a genomic locus or landing site of a host cell (e g., HEK293 cell) comprising the complementary recognition site.

[00171] In some embodiments, the disclosure further provides a host cell comprising one or more recombinant polynucleotides described herein. In some embodiments, the one or more recombinant polynucleotides described herein are stably integrated into the host cell genome (c.g., the host cell nuclear genome). In some embodiments, the host cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell. Vero cell, or PerC6 cell. In some embodiments, the host cell is a HEK293 cell or a HEK293 derived cell.

Polynucleotide vectors

[00172] In some embodiments, the disclosure provides a polynucleotide vector comprising a recombinant polynucleotide described herein. In some embodiments, the polynucleotide vector comprises a recombinant polynucleotide encoding one or more helper functions, one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the polynucleotide vector comprises a recombinant polynucleotide encoding one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the polynucleotide vector comprises a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene. In some embodiments, the one or more AAV rep genes encode a Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, Rep40 polypeptide or any combination thereof. In some embodiments, the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the AAV cap gene further encodes an assemblyactivating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP). In some embodiments, the polynucleotide vector comprises a recombinant packaging polynucleotide described herein.

[00173] In some embodiments, a polynucleotide vector described herein is a bacterial plasmid. In some embodiments, the polynucleotide vector comprises a bacterial replication origin capable of propagating the plasmid in a bacterial host cell, e.g.. E. coli host cell. In some embodiments, the bacterial replication origin is a ColE 1 origin.

[00174] In some embodiments, a polynucleotide vector described herein comprises a selectable marker gene. In some embodiments, the selectable marker gene is a drug resistance gene. In some embodiments, the selectable marker gene is a kanamycin resistance gene. In some embodiments, the selectable marker gene is an ampicillin resistance gene. In some embodiments, the selectable marker gene is a puromycin resistance gene. In some embodiments, the selectable marker gene is suitable for the selection of stable transfectants in mammalian host cells.

[00175] In some embodiments, a polynucleotide vector described herein comprises a bacterial replication origin and a selectable marker gene.

[00176] In some embodiments, a polynucleotide vector described herein comprises one or more elements capable of maintaining expression of stably integrated transgenes in eukaryotic cells. In some embodiments, a polynucleotide vector described herein comprises one or more cHS4 insulator elements. In some embodiments, a cHS4 insulator element comprises the nucleotide sequence of SEQ ID NO: 40 or 41. [00177] In some embodiments, a polynucleotide vector described herein comprises a constitutive eukaryotic promoter operably linked to a polynucleotide encoding a doxycycline-responsive transactivator protein. In some embodiments, the promoter is an hPKG promoter, CMV promoter or SV40 promoter. In some embodiments, the doxycycline-responsive transactivator protein comprises the amino acid sequence of SEQ ID NO: 92.

[00178] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the inducible gene expressing an adenovirus E4 polypeptide 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: 23-28 or 29, tire inducible gene expressing an adenovirus E2A DNA binding protein (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: 9, and the VA RNA 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 to SEQ ID NO: 30. In some embodiments, tire inducible gene expressing an adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 23- 28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9, and the VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9, and the VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9, and the VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9, and the VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide 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: 23. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide 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: 28.

[00179] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide vector 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: 33-38 or 39. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 33-38 or 39. In some embodiments, tire polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 33-38 or 39. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 33-38 or 39. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 33-38 or 39.

[00180] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide vector 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: 33. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 33. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 33. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 33. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 33. [00181] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide vector 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: 38. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 38. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 38. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 38. In some embodiments, tire polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 38.

[00182] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide vector 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: 93-98 or 99. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 93-98 or 99. In some embodiments, tire polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 93-98 or 99.

[00183] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide vector 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: 93. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 93. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 93. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 93. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 93.

[00184] In some embodiments, a polynucleotide vector described herein comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide vector 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: 98. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 98. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 98. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 98. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 98.

[00185] In some embodiments, a polynucleotide vector described herein comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, and a gene expressing an L4 22K/33K polypeptide, wherein tire polynucleotide vector 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: 48. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 48.

[00186] In some embodiments, a polynucleotide vector described herein comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, a gene expressing an L4 22K/33K polypeptide, and an inducible gene expressing AAV Rep40 and Rep52 polypeptides, wherein the polynucleotide vector 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: 75. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 75. [00187] In some embodiments, a polynucleotide vector described herein comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, a gene expressing an L4 22K/33K polypeptide, and an inducible gene expressing AAV Rep40 and Rep52 polypeptides, wherein the polynucleotide vector 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: 100. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 100. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 100. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 100. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 100.

[00188] In some embodiments, a polynucleotide vector described herein comprises an inducible gene with an intron expressing AAV Rep polypeptides and an inducible gene expressing AAV Cap polypeptides, wherein the polynucleotide vector 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: 77-81 or 82. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 77-81 or 82. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 77-81 or 82. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 77-81 or 82. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 77-81 or 82.

[00189] In some embodiments, a polynucleotide vector described herein comprises an inducible gene with an intron expressing AAV Rep polypeptides and an inducible gene expressing AAV Cap polypeptides, wherein the polynucleotide vector 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: 103-107 or 108. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 103-107 or 108. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 103-107 or 108. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 103-107 or 108. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 103-107 or 108.

[00190] In some embodiments, a polynucleotide vector described herein comprises an inducible gene expressing AAV Cap polypeptides and AAV Rep78/68 polypeptides, wherein the polynucleotide vector 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: 73. 101 or 102. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 73, 101 or 102. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 73, 101 or 102. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 73, 101 or 102. In some embodiments, the polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 73, 101 or 102.

[00191] In some embodiments, a polynucleotide vector described herein comprises an inducible gene expressing AAV Cap polypeptides and AAV Rep78/68 polypeptides, wherein the polynucleotide vector 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: 83- 90 or 91. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 83-90 or 91. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 83-90 or 91. In some embodiments, the polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 83-90 or 91. In some embodiments, tire polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 83-90 or 91.

[00192] In some embodiments, a polynucleotide vector described herein comprises a packaging polynucleotide described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding Original iHelper or iHelper#l-9 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding Original iHelper or iHelper#l-9 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#6 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#6 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises SEQ ID NO: 48 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises SEQ ID NO: 48 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHclper#7 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelpcr#8 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises flanking recognition sites for an integrase or recombinase such that the packaging polynucleotide can be integrated into a genomic landing site of a host cell (e.g., HEK293 cell) comprising the complementary recognition site.

RECOMBINANT HOST CELLS

[00193] In some embodiments, the disclosure provides a host cell comprising a recombinant polynucleotide or a polynucleotide vector described herein. In some embodiments, the host cell is a prokary otic cell capable of propagating a recombinant polynucleotide or a polynucleotide vector described herein. In some embodiments, the prokary otic host cell is a bacterial cell. In some embodiments, the prokaryotic host cell is E. coli. In some embodiments, the host cell is a eukary otic cell capable of producing recombinant AAV particles. In some embodiment, a eukaryotic cell capable of producing recombinant AAV particles comprises a stably^r integrated recombinant polynucleotide described herein or a stably integrated polynucleotide vector described herein. In some embodiments, the eukaryotic host cell is a mammalian cell. In some embodiments, the eukaryotic host cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell. HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00194] In some embodiments, a host cell described herein comprises one or more recombinant polynucleotide described herein. In some embodiments, host cell comprises one or more stably integrated recombinant polynucleotide described herein. In some embodiments, the recombinant polynucleotide encodes one or more helper functions, one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the recombinant polynucleotide described herein encodes one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the recombinant polynucleotide described herein encodes one or more AAV rep genes and an AAV cap gene. In some embodiments, the one or more AAV rep genes encode a Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, Rep40 polypeptide or any combination thereof. In some embodiments, the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the AAV cap gene further encodes an assembly-activating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP). In some embodiments, the recombinant polynucleotide is a packaging polynucleotide described herein. In some embodiments, one or more recombinant polynucleotides described herein are transiently transfected into a host cell. In some embodiments, one or more recombinant polynucleotides described herein are stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the host cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the host cell is a HEK293 cell or a HEK293 derived cell.

[00195] In some embodiments, a host cell described herein comprises a polynucleotide vector described herein. In some embodiments, the polynucleotide vector comprises a recombinant polynucleotide encoding one or more helper functions, one or more AAV rep genes, an AAV cap gene, or any combination thereof. In some embodiments, the polynucleotide vector comprises a recombinant polynucleotide encoding one or more helper functions. In some embodiments, the one or more helper functions comprise a gene expressing an adenovirus E2A DNA binding protein (DBP), a gene expressing an adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide), an adenovirus VA RNA gene or any combinations thereof. In some embodiments, the gene expressing the adenovirus E2A DNA binding protein (DBP) and/or the gene expressing the adenovirus E4 polypeptide (e.g., an E4orf6 or E4orf6-6/7 polypeptide) is an inducible gene. In some embodiments, the one or more helper functions further comprise a gene expressing an adenovirus L4 22K/33K polypeptide. In some embodiments, the polynucleotide vector comprises a recombinant polynucleotide encoding one or more AAV rep genes and an AAV cap gene. In some embodiments, the one or more AAV rep genes encode a Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, Rep40 polypeptide or any combination thereof. In some embodiments, the AAV cap gene encodes a VP1, VP2 and VP3 capsid polypeptide. In some embodiments, one or more AAV rep genes and/or AAV cap gene is an inducible gene. In some embodiments, the AAV cap gene further encodes an assembly-activating protein (AAP) polypeptide. In some embodiments, the AAV cap gene further encodes a membrane-associated assembly protein (mAAP). In some embodiments, a polynucleotide vector described herein is transiently transfected into a host cell. In some embodiments, a polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the host cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell. SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the host cell is a HEK293 cell or a HEK293 derived cell. In some embodiments, the polynucleotide vector is a bacterial plasmid.

[00196] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the inducible gene expressing an adenovirus E4 polypeptide 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: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (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: 9, and the VA RNA 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 to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 9, and the VA RNA gene comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 9, and the VA RNA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 9, and the VA RNA gene comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide comprises the nucleotide sequence of SEQ ID NO: 23-28 or 29, the inducible gene expressing an adenovirus E2A DNA binding protein (DBP) comprises the nucleotide sequence of SEQ ID NO: 9, and the VA RNA gene comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide 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: 23. In some embodiments, the inducible gene expressing an adenovirus E4 polypeptide 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: 28. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00197] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector 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: 33-38 or 39. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 33-38 or 39. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 33-38 or 39. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 33-38 or 39. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 33-38 or 39. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g.. SEQ ID NO: 92).

[00198] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector 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: 33. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 33. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 33. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 33. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, tire host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., tire host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00199] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector 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: 38. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 38. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 38. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 38. In some embodiments, the polynucleotide or polynucleotide vector comprises tire polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e g., SEQ ID NO: 92).

[00200] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector 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: 93-98 or 99. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 93-98 or 99. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a EIEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00201] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide or polynucleotide vector comprises the polynucleotide or polynucleotide vector 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: 93. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 93. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 93. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 93. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00202] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises, in 5' to 3' direction, an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, wherein the polynucleotide or polynucleotide vector 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: 98. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 98. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 98. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 98. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 98. In some embodiments, tire host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., tire host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00203] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, and a gene expressing an L4 22K/33K polypeptide, wherein the polynucleotide or polynucleotide vector 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: 48. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 48. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a EIEK293 cell. HEK293 derived cell, CEIO cell, or CHO derived cell. In some embodiments, tire polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00204] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, a gene expressing an L4 22K/33K polypeptide, and an inducible gene expressing AAV Rep40 and Rep52 polypeptides, wherein the polynucleotide or polynucleotide vector 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: 75. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 75. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 75. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, EIEK293 derived cell, CHO cell, or CEIO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00205] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, a gene expressing an L4 22K/33K polypeptide, and an inducible gene expressing AAV Rep40 and Rep52 polypeptides, wherein the polynucleotide or polynucleotide vector 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: 100. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 100. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 100. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 100. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 100. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline -responsive transactivator protein (e.g., SEQ ID NO: 92).

[00206] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene with an intron expressing AAV Rep polypeptides and an inducible gene expressing AAV Cap polypeptides, wherein the polynucleotide or polynucleotide vector 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: 77-81 or 82. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 77-81 or 82. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 77-81 or 82. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 77-81 or 82. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 77-81 or 82. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g.. the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00207] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene with an intron expressing AAV Rep polypeptides and an inducible gene expressing AAV Cap polypeptides, wherein the polynucleotide or polynucleotide vector 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: 103-107 or 108. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 103-107 or 108. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 103-107 or 108. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 103-107 or 108. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 103-107 or 108. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell. CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00208] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene expressing AAV Cap polypeptides and AAV Rep78/68 polypeptides, wherein the polynucleotide or polynucleotide vector 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: 73, 101 or 102. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 73, 101 or 102. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 73. 101 or 102. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 73, 101 or 102. In some embodiments, the polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 73, 101 or 102. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a EIEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00209] In some embodiments, a recombinant host cell comprises a polynucleotide described herein or a polynucleotide vector described herein, wherein the polynucleotide or polynucleotide vector comprises an inducible gene expressing AAV Cap polypeptides and AAV Rep78/68 polypeptides, wherein the polynucleotide or polynucleotide vector 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: 83-90 or 91. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 83-90 or 91. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 83-90 or 91. In some embodiments, the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 83-90 or 91. In some embodiments, tire polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 83-90 or 91. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g.. SEQ ID NO: 92).

[00210] In some embodiments, a recombinant host cell comprises a first and second polynucleotide or polynucleotide vector described herein, wherein (i) the first polynucleotide or polynucleotide vector comprises an inducible gene expressing AAV Cap polypeptides and AAV Rep78/68 polypeptides, (ii) the second polynucleotide or polynucleotide vector comprises an inducible gene expressing an adenovirus E4 polypeptide and adenovirus E2A DNA binding protein (DBP), an adenovirus VA RNA gene, a gene expressing an L4 22K/33K polypeptide, and an inducible gene expressing AAV Rep40 and Rep52 polypeptides, (iii) the first polynucleotide or polynucleotide vector 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: 73, and (iv) the second polynucleotide or polynucleotide vector 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: 75. In some embodiments, the first polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 73 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 75. In some embodiments, the first polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 73 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 75. In some embodiments, first the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 73 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 75. In some embodiments, the first polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 73 and the second polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 75. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the first and second polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., tire host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e g., SEQ ID NO: 92). [00211] In some embodiments, a recombinant host cell comprises a first and second polynucleotide or polynucleotide vector described herein, wherein (i) the first polynucleotide or polynucleotide vector comprises inducible gene with an intron expressing AAV Rep polypeptides and an inducible gene expressing AAV Cap polypeptides, (ii) the second polynucleotide or polynucleotide vector comprises an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, (iii) the first polynucleotide or polynucleotide vector 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: 77-81 or 82, and (iv) the second polynucleotide or polynucleotide vector 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: 93- 98 or 99. In some embodiments, the first polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 77-81 or 82 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the first polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 77-81 or 82 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 93-98 or 99. In some embodiments, first the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 77-81 or 82 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 93-98 or 99. In some embodiments, the first polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 77-81 or 82 and tire second polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 93-98 or 99. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the first and second polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00212] In some embodiments, a recombinant host cell comprises a first and second polynucleotide or polynucleotide vector described herein, wherein (i) the first polynucleotide or polynucleotide vector comprises inducible gene with an intron expressing AAV Rep polypeptides and an inducible gene expressing AAV Cap polypeptides, (ii) the second polynucleotide or polynucleotide vector comprises an inducible gene expressing an adenovirus E4 polypeptide, an inducible gene expressing an adenovirus E2A DNA binding protein (DBP), and an adenovirus VA RNA gene, (iii) the first polynucleotide or polynucleotide vector 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: 80, 81 or 82, and (iv) the second polynucleotide or polynucleotide vector 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: 93 or 98. In some embodiments, tire first polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 80, 81 or 82 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 93 or 98. In some embodiments, the first polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 80, 81 or 82 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 93 or 98. In some embodiments, first the polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 80, 81 or 82 and the second polynucleotide or polynucleotide vector comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 93 or 98. In some embodiments, the first polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 80, 81 or 82 and the second polynucleotide or polynucleotide vector comprises the nucleotide sequence of SEQ ID NO: 93 or 98. In some embodiments, the first polynucleotide or polynucleotide vector 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: 80. In some embodiments, the first polynucleotide or polynucleotide vector 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: 81. In some embodiments, the first polynucleotide or polynucleotide vector 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: 82. In some embodiments, the second polynucleotide or polynucleotide vector 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: 93. In some embodiments, the second polynucleotide or polynucleotide vector 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: 98. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the first and second polynucleotide or polynucleotide vector described herein is stably integrated into the host cell genome (e.g.. the host cell nuclear genome). In some embodiments, the eukaryotic host cell comprises a doxycycline-responsive transactivator protein (e.g., SEQ ID NO: 92).

[00213] In some embodiments, a host cell described herein comprises a packaging polynucleotide described herein. In some embodiments, the packaging polynucleotide is stably integrated into the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the packaging polynucleotide is stably integrated into the host cell genome (e.g., the host cell nuclear genome) at a safe harbor locus. In some embodiments, the packaging polynucleotide has been stably integrated into tire host cell genome by nuclease, recombinase or integrase mediated integration. In some embodiments, the packaging polynucleotide has been stably integrated into the host cell genome at a safe harbor locus by nuclease, recombinase or integrase mediated integration. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding Original iHelper or iHelper# 1-9 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding Original iHelper or iHelper# 1-9 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#6 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6. iTrans#7. iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#6 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding iTrans#5, iTrans#10, iTrans#6, iTrans#7, iTrans#8, iTrans#7TetCap described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding a polynucleotide encoding 3.4-3.7 or 5.0-5.4 described herein. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein tire first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises SEQ ID NO: 48 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding one or more helper functions, wherein the first polynucleotide comprises SEQ ID NO: 48 and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#7 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82. In some embodiments, the packaging polynucleotide comprises a first polynucleotide encoding iHelper#8 described herein and a second polynucleotide encoding one or more AAV rep genes and AAV cap gene, wherein the second polynucleotide comprises any one of SEQ ID NO: 83-91. In some embodiments, the packaging polynucleotide comprises flanking recognition sites for an integrase or recombinase such that tire packaging polynucleotide can be integrated into a genomic landing site of a host cell (e.g., HEK293 cell) comprising the complementary recognition site. In some embodiments, the host cell further comprises a cis polynucleotide encoding a recombinant AAV genome. In some embodiments, the cis polynucleotide is stably integrated int the host cell genome (e.g., the host cell nuclear genome). In some embodiments, the cis polynucleotide comprises at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product (e.g., therapeutic gene product) in a target cell. 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 gene product is a dystrophin or a microdystrophin. In some embodiments, the gene product is a microRNA, snRNA or an antisense RNA. In some embodiments, the cis polynucleotide comprises in a 5 'to 3’ direction a (a) 5’ AAV2 inverted tenninal repeat sequence (5‘ ITR), (b) a promoter (e.g., a tissue-specific promoter) operably linked to (c) a transgene (e.g., therapeutic transgene) coding region, and (d) a 3’ AAV2 inverted terminal repeat sequence (5’ ITR). In some embodiments, the cis polynucleotide comprises flanking recognition sites for an integrase or recombinase such that the cis polynucleotide can be integrated into a genomic landing site of a host cell (e.g., HEK293 cell) comprising the complementary recognition site. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a HEK293 cell, HEK293 derived cell, CHO cell, or CHO derived cell. In some embodiments, the eukaryotic host cell comprises integration sites/landing pads for insertion of multiple copies of the packaging polynucleotide and/or cis construct. In some embodiments, the eukaryotic host cell comprises a doxycycline -responsive transactivator protein (e.g., SEQ ID NO: 92).

[00214] In some embodiments, the disclosure provides a method of producing a recombinant polynucleotide described herein or a polynucleotide vector described herein comprising incubating a host cell described herein under suitable conditions to produce the recombinant polynucleotide or a polynucleotide vector. In some embodiments, the host cell is a prokaryotic cell capable of propagating a polynucleotide vector described herein. In some embodiments, the prokaryotic host cell is a bacterial cell. In some embodiments, the prokary otic host cell is E. coli.

METHODS OF MAKING STABLE HOST CELLS

[00215] Methods of making host cells that stably express a recombinant gene in the host cell are known and may be performed by integration of transgene(s) into a genomic locus such as a safe harbor locus. Safe harbor loci are regions of genomic DNA within the host cells that accept integration of an exogenous gene while ensuring proper function of the exogenous genetic material and no disruption or alteration to the host genome. Recent advances in gene editing and gene targeting technologies allow for efficient methods of making stable host cells.

[00216] Genomic loci that are known as safe harbor sites include, but are not limited to, tire adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR ) gene, the Rosa26 locus or a species-specific ortholog thereof, Region Optimal for Gene Insertions 1 (Rogil) and Region Optimal for Gene Insertions 2 (Rogi2) (Papapetrou and Schlambach, Mol. Ther. vol. 24 no. 4, 678-684 apr. 2016; Aznauryan, 2022, Cell Reports Methods 2, 100154). Targeted integration into these and other loci may be accomplished by various genome-editing strategics based on common nuclease -based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR/Cas9 platforms (Maeder and Gersbach, Mol. Ther. Volume 24, Issue 3, March 2016, Pages 430-446). Expression cassettes and polynucleotides described herein are made into targeting vectors that comprise a 5’ homology arm targeting a 5’ target sequence at a target genomic locus and a 3’ homology arm targeting a 3’ targeting sequence at the target genomic locus. The targeting vector is transfected into the host cell of interest such that the expression cassette integrates at the target genomic locus of the cell. In some methods, in the presence of a nuclease agent the targeting vector recombinase with the target genomic locus. Host cell clones with expression cassettes and polynucleotides as described “knocked-in” or targeted to genomic loci enables selection of stable clones that produce rAAV particles (vectors) with high titer and suitable full /empty ratio of rAAV particles following cell suspension culture.

METHODS OF PRODUCING A RECOMBINANT VIRAL PARTICLE

[00217] In one aspect, the disclosure provides a method of producing recombinant adeno- associated virus (rAAV) particles in a eukaryotic host cell by using a recombinant polynucleotide, polynucleotide vector or host cell described herein. In some embodiments, the method further comprises recovering the rAAV particles.

[00218] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated vims (rAAV) particles comprising (a) providing a cell culture comprising the recombinant cell described herein, (b) introducing into the cell (i) a polynucleotide encoding an AAV capsid protein; (ii) a polynucleotide encoding a functional rep gene: and (iii) a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (1TR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of tire gene product in a target cell, and (c) maintaining the cell under conditions that allow production of the rAAV particles, wherein the recombinant cell described herein comprises a stably integrated polynucleotide described herein encoding an adenovirus E2A DNA binding protein (DBP), an adenovirus E4 polypeptide and an adenovirus VA RNA gene. In some embodiments, the recombinant cell is a HEK293 cell, HEK293 derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the recombinant cell is a HEK293 cell or HEK293 derived cell. In some embodiments, the recombinant cell culture is a suspension culture. In some embodiments, the method further comprises recovering the rAAV particles. In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. 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 gene product is a dystrophin or a microdystrophin. In some embodiments, the gene product is amicroRNA, snRNA or an antisense RNA.

[00219] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated vims (rAAV) particles comprising (a) providing a cell culture comprising the recombinant cell described herein; (b) introducing into the cell (i) a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; and (ii) a polynucleotide comprising sufficient helper functions to permit packaging of the genome into tire AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid, and (c) maintaining the cell under conditions that allow production of the rAAV particles, wherein the cell described herein comprises a stably integrated polynucleotide described herein encoding one or more AAV rep genes and an AAV cap gene. In some embodiments, the recombinant cell is a HEK293 cell, HEK293 derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the recombinant cell is a HEK293 cell or HEK293 derived cell. In some embodiments, the recombinant cell culture is a suspension culture. In some embodiments, the method further comprises recovering the rAAV particles. In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. 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 gene product is a dystrophin or a microdystrophin. In some embodiments, tire gene product is amicroRNA. snRNA or an antisense RNA.

[00220] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated vims (rAAV) particles comprising (a) providing a cell culture comprising a recombinant cell described herein; (b) introducing into the cell a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; and (c) maintaining the cell under conditions that allow production of the rAAV particles, wherein the recombinant cell described herein comprises (i) a first stably integrated polynucleotide described herein encoding an adenovirus E2A DNA binding protein (DBP), an adenovirus E4 polypeptide and an adenovirus VA RNA gene and (ii) a second stably- integrated polynucleotide described herein encoding one or more AAV rep genes and an AAV cap gene. In some embodiments, the recombinant cell is a HEK.293 cell, HEK293 derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the recombinant cell is a HEK293 cell or HEK293 derived cell. In some embodiments, the recombinant cell culture is a suspension culture. In some embodiments, the method further comprises recovering the rAAV particles. In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. In some embodiments, the gene product is a polypeptide or a double stranded RNA molecule. In some embodiments, tire gene product is a polypeptide. In some embodiments, the gene product is a dystrophin or a microdystrophin. In some embodiments, tire gene product is amicroRNA. snRNA or an antisense RNA.

[00221] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated vims (rAAV) particles comprising (a) providing a cell culture comprising the recombinant cell described herein; and (b) maintaining the cell under conditions that allow production of the rAAV particles, wherein tire recombinant cell described herein comprises (i) a first stably integrated polynucleotide described herein encoding an adenovirus E2A DNA binding protein (DBP), an adenovirus E4 polypeptide and an adenovirus VA RNA gene, (ii) a second stably integrated polynucleotide described herein encoding one or more AAV rep genes and an AAV cap gene, (iii) and a third polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell. In some embodiments, the third polynucleotide is stably integrated. In some embodiments, the recombinant cell is a HEK293 cell, HEK293 derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the recombinant cell is a HEK293 cell or HEK293 derived cell. In some embodiments, the recombinant cell culture is a suspension culture. In some embodiments, the method further comprises recovering the rAAV particles. In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. 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 gene product is a dystrophin or a microdystrophin. In some embodiments, the gene product is a microRNA, snRNA or an antisense RNA.

[00222] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles comprising culturing a cell capable of producing the rAAV particles, wherein the cell comprises (i) a polynucleotide encoding an AAV capsid protein; (ii) a polynucleotide encoding a functional rep gene; (iii) a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; and (iv) one or more polynucleotides comprising sufficient helper functions to permit packaging of the genome into the AAV capsid protein under conditions which permit packaging of the genome into tire AAV capsid, wherein the recombinant polynucleotide described herein or a polynucleotide vector described herein comprise a recombinant polynucleotide encoding one or more helper functions, an AAV rep gene and an AAV cap gene, and optionally a recombinant AAV viral genome comprising a nucleic acid sequence encoding a gene product. In some embodiments, the one or more helper functions comprise a nucleotide sequence encoding tire adenovirus E2A DBP, a nucleotide sequence encoding the adenovirus E4 polypeptide and a nucleotide sequence encoding the adenovirus VA RNA I. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises the E4 ORF6. In some embodiments, the nucleotide sequence encoding an adenovirus VA RNA I encodes an adenovirus VA RNA I and VA RNA II. In some embodiments, the method further comprises recovering the rAAV particles. In some embodiments, the cell comprises (i) one polynucleotide described 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) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene and (ii) one polynucleotide encoding the rAAV genome to be packaged. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises the E4 ORF6. In some embodiments, the cell comprises one polynucleotide described herein comprising (a) a nucleotide sequence encoding an adenovirus E2A 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 AAV viral 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 the E4 ORF6. 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.rhlO, 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 such as, AAV.rhlO, 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, tire cell culture comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume of between about 400 liters and about 5,000 liters.

[00223] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell a first polynucleotide disclosed here 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) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene and a second polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell, and (c) maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises the E4 ORF6. In some embodiments, the disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell a polynucleotide vector disclosed here 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) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene and a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell, and (c) maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises the E4 ORF6. In some embodiments, the nucleotide sequence encoding an adenovirus VA RNA I encodes an 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 tire group consisting of AAV.rh8, AAV.rhlO, 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 such as, AAV.rhlO, 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 comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume of between about 400 liters and about 5.000 liters.

[00224] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles comprising (a) providing a cell culture comprising a cell: (b) introducing into the cell a polynucleotide disclosed herein comprising (i) a nucleotide sequence encoding an adenovirus E2A DBP, (ii) a nucleotide sequence encoding an adenovirus E4 polypeptide, (iii) a nucleotide sequence encoding an adenovirus VA RNA I, (iv) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene, and (v) a nucleotide sequence encoding a recombinant AAV viral genome comprising a nucleic acid sequence encoding a gene product, and (c) maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises the E4 ORF6. In some embodiments, the disclosure provides a method of producing recombinant adcno-associatcd virus (rAAV) particles comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell a polynucleotide vector disclosed here comprising (i) a nucleotide sequence encoding an adenovirus E2A DBP, (ii) a nucleotide sequence encoding an adenovirus E4 polypeptide, (iii) a nucleotide sequence encoding an adenovirus VA RNA I, (iv) a nucleotide sequence encoding an AAV rep gene and an AAV cap gene, and (v) a nucleotide sequence encoding a recombinant AAV viral genome comprising a nucleic acid sequence encoding a gene product, and (c) maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the adenovirus E4 polypeptide comprises E4 ORF6 and ORF7. In some embodiments, the adenovirus E4 polypeptide comprises the E4 ORF6. In some embodiments, the nucleotide sequence encoding an adenovirus VA RNA I encodes an 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 tire group consisting of AAV.rh8, AAV.rhlO, 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 such as, AAV.rhlO, 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 comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume of between about 400 liters and about 5.000 liters.

[00225] In some embodiments, the disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles comprising (a) providing a cell culture comprising a recombinant host cell described herein; and (b) maintaining the cell under conditions that allow production of the rAAV particles, wherein the recombinant host cell described comprises (i) a stably integrated packaging polynucleotide described herein, and (ii) a stably integrated cis polynucleotide described herein encoding a genome comprising at least one AAV inverted terminal repeat (ITR) and a non- AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell. In some embodiments, the recombinant cell is a HEK293 cell, HEK293 derived cell, CHO cell, CEIO derived cell, EleLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the recombinant cell is a HEK293 cell or HEK293 derived cell. In some embodiments, the recombinant cell culture is a suspension culture. In some embodiments, the method further comprises recovering the rAAV particles. In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. 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 gene product is a dystrophin or a microdystrophin. In some embodiments, the gene product is a microRNA, snRNA or an antisense RNA.

[00226] In some embodiments, a method described herein comprises introducing into the cell a polynucleotide encoding an AAV capsid protein and a functional rep gene.

[00227] In some embodiments, the introducing of the one or more polynucleotides and/or the one or more polynucleotide vectors into the cell is by transfection. [00228] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell. In some embodiments, the cell is a HEK293 cell.

[00229] In some embodiments, tire cell culture is a suspension culture or an adherent culture. In some embodiments, the cell culture is a suspension culture.

[00230] In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters.

[00231] In some embodiments, a method described herein produces more rAAV particles measured as GC/ml than a 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 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 than the reference method. In some embodiments, the method produces at least about three times as many rAAV particles measured as GC/ml than the reference method. In some embodiments, the method produces at least about four times as many rAAV particles measured as GC/ml than the reference method.

[00232] In some embodiments, the method produces a population of rAAV particles comprising more full capsids than a reference method.

[00233] In some embodiments, the rAAV particles comprise a capsid protein of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, 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 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8, AAV9, AAV.rhlO. AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, or AAV.hu37 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 serotype. In some embodiments, tire rAAV particles comprise a capsid protein of the AAV9 serotype.

[00234] In some embodiments, tire rAAV particle comprises 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 antigen-binding fragment thereof, fusion protein, Fc-fusion polypeptide, immunoadhesin, immunoglobulin, engineered protein, protein fragment or enzyme. In some embodiments, the transgcnc comprises a regulatory’ clement operatively connected to a polynucleotide encoding the gene product.

[00235] 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 nonmembrane associated splice variant of VEGF receptor 1 (sFlt-1). In some embodiments, the gene product is an gamma-sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid isomcrohydrolasc (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated 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), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1). arylsulfatase B (ARSB), N-acetyl- alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), betagalactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4). protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR). or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgGl) Fc fusion. In some embodiments, the gene product is a dystrophin or a microdystrophin. In some embodiments, the gene product is a microRNA, snRNA or antisense RNA.

[00236] In some embodiments, a method described herein increases production of rAAV particles while maintaining or improving the quality attributes of the rAAV particles and compositions comprising thereof. In some embodiments, the quality of rAAV particles and compositions comprising thereof is assessed by determining the concentration of rAAV particles (e.g., GC/ml). the percentage of particles comprising a copy of the rAAV genome; the ratio of particles without a genome, infectivity of the rAAV particles, stability of rAAV particles, concentration of residual host cell proteins, or concentration of residual host cell nucleic acids (c.g., host cell genomic DNA, plasmid encoding rep and cap genes, plasmid encoding helper functions, plasmid encoding rAAV genome). In some embodiments, the quality of rAAV particles produced by a method described herein or compositions comprising thereof is the same as that of rAAV particles or compositions produced by a reference method. In some embodiments, the quality of rAAV particles produced by a method described herein or compositions comprising thereof is better than the quality of rAAV particles or compositions produced by a reference method.

[00237] Numerous cell culture based systems are known in the art for production of rAAV particles, any of which can be used to practice a method described herein. rAAV production cultures for the production of rAAV virus particles require; (1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), or mammalian cell lines such as Vero, CHO cells or CHO- derived cells; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production. [00238] A skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. Tire phrase “adenovirus helper functions" refers to a number of viral helper genes expressed in a cell (as RNA or protein) such that the AAV grows efficiently in the cell. The skilled artisan understands that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g., the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments of a method described herein, AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.

[00239] Molecular biology techniques to develop plasmid or viral vectors encoding the AAV rep and cap genes, helper genes, and/or rAAV genome are commonly known in the art. In some embodiments, AAV rep and cap genes arc encoded by one plasmid vector. In some embodiments, AAV helper genes (e.g.. adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one plasmid vector. In some embodiments, the Ela gene or Elb gene is stably- expressed by the host cell, and tire remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector. In some embodiments, one or more helper genes arc stably- expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, AAV rep and cap genes are encoded by one viral vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector. In some embodiments, tire Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene. E2a gene, and VA gene are introduced into the cell by transfection by one viral 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 transfection by one viral vector. In some embodiments, the AAV rep and cap genes, the adenovirus helper functions necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, a method described herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding tire cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g.. adenovirus Ela gene. Elb gene, E4 gene. E2a gene, and VA gene), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is an AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, 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.rhlO. 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 comprises a gene of interest flanked by AAV ITRs. In some embodiments, the AAV ITRs are from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, 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 or other AAV serotype.

[00240] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged. In some embodiments of a method described herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding helper genes can be used. In some embodiments, a mixture of the three vectors is co-transfected into a cell.

[00241] In some embodiments, a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors.

[00242] In some embodiments, one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells. In some embodiments, the cell constitutively expresses rep and/or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses El a. In some embodiments, the cell comprises a stable transgene encoding the rAAV genome.

[00243] In some embodiments, AAV rep, cap, and helper genes (e.g., Ela gene, Elb gene, E4 gene, E2a gene, or VA gene) can be of any AAV serotype. Similarly, AAV ITRs can also be of any AAV serotype. For example, in some embodiments, AAV ITRs are from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, 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.HSCI6 or other AAV serotypes (e g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV cap gene is from AAV9 or AAV8 cap gene. In some embodiments, an AAV cap gene is from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11. AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, 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.HSC 16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV rep and cap genes for the production of a rAAV particle are from different serotypes. For example, the rep gene is from AAV2 whereas the cap gene is from AAV9.

[00244] Any suitable media known in the art can be used for the production of recombinant virus particles (e.g., rAAV particles) according to a method described herein. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety. In some embodiments, the medium comprises Dynamis™ Medium, FreeStyle™ 293 Expression Medium, or Expi293™ Expression Medium from Invitrogen/ ThermoFisher. In some embodiments, a method described herein uses a cell culture comprising a serum-free medium, an animal -component free medium, or a chemically defined medium. In some embodiments, the medium is an animal-component free medium. In some embodiments, the medium comprises serum. In some embodiments, the medium comprises fetal bovine serum. In some embodiments, the medium is a glutamine-free medium. In some embodiments, the medium comprises glutamine. In some embodiments, the medium is supplemented with one or more of nutrients, salts, buffering agents, and additives (e.g., antifoam agent). In some embodiments, the medium is supplemented with glutamine. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is supplemented with fetal bovine serum. In some embodiments, the medium is supplemented with poloxamer, e.g., Kolliphor® P 188 Bio. In some embodiments, a medium is a base medium. In some embodiments, the medium is a feed medium.

[00245] Recombinant virus (e.g., rAAV) production cultures can routinely be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, virus production cultures include suspension-adapted host cells such as HcLa 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 and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank biorcactors, and disposable systems such as the Wave bag system. Numerous suspension cultures are known in the art for production of rAAV particles, including for example, the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No. 20120122155, each of which is incorporated herein by reference in its entirety.

[00246] Any cell or cell line that is 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, a method of producing recombinant virus particles (e.g., rAAV particles) or increasing the production of recombinant virus particles (e.g., a rAAV particles) described herein uses 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, a method described herein uses mammalian cells. In some embodiments, a method described herein uses insect cells, e.g.. SF-9 cells. In some embodiments, a method described herein uses cells adapted for growth in suspension culture. In some embodiments, a method described herein uses HEK293 cells adapted for growth in suspension culture.

[00247] In some embodiments, a cell culture described herein is a suspension culture. In some embodiments, a large scale suspension cell culture described herein comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, a cell culture described herein comprises a serum-free medium, an animal-component free medium, or a chemically defined medium. In some embodiments, a cell culture described herein comprises a serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaker flask, a spinner flask, a cell bag, or a bioreactor.

[00248] In some embodiments, a cell culture described herein comprises a serum-free medium, an animal -component free medium, or a chemically defined medium. In some embodiments, a cell culture described herein comprises a serum-free medium.

[00249] In some embodiments, a large scale suspension culture cell culture described herein comprises a high density cell culture. In some embodiments, the culture has a total cell density of between about lxl0E+06 cells/ml and about 30xl0E+06 cells/ml. In some embodiments, more than about 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), Vcro cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells.

[00250] Methods described herein can be used in the production of rAAV particles comprising a capsid protein from any AAV capsid serotype. In some embodiments, the rAAV particles comprise a capsid protein 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.rhlO, 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 comprise a capsid protein that is a derivative, modification, or pseudotype of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, 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 capsid protein.

[00251] 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 an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

[00252] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles comprise a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174. AAV.RHM4-1, AAV.hu32, and AAV.hu37.

[00253] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 or AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more 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, to the VP1, VP2 and/or VP3 sequence of AAV8 capsid protein.

[00254] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV9 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that has an AAV9 capsid protein at least 80% or more 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, to the VP1, VP2 and/or VP3 sequence of AAV9 capsid protein. [00255] In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, 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% identity, to the VP1, VP2 and/or VP3 sequence of AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, or AAV.7m8 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, 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% identity, to the VP1, VP2 and/or VP3 sequence of an AAV capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37.

[00256] In additional embodiments, the rAAV particles comprise a mosaic capsid. In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle. In additional embodiments, the rAAV particles comprise a capsid containing a capsid protein chimera of two or more AAV capsid serotypes.

RAAV PARTICLES

[00257] The provided compositions and methods are suitable for use in the production of any isolated recombinant AAV particles. As such, the rAAV can be of any serotype, modification, or derivative, known in the art, or any combination thereof (e.g., a population of rAAV particles that comprises two or more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles) known in the art. In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, 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 or other rAAV particles, or combinations of two or more thereof.

[00258] In some embodiments, rAAV particles have a capsid protein from an AAV serotype selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8. AAV.rhlO, 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.HSC 16 or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more 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, to e.g., VP 1. VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, 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.HSC3, AAV.HSC4, AAV.HSC5. AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9. AAV.HSC 10 , AAV.HSC11. AAV.HSC12, AAV.HSC13, AAV.HSC14. AAV.HSC15, or AAV.HSC16. [00259] In some embodiments, rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, 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 1, 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.HSC 14, AAV.HSC 15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more 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, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, 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. [00260] In some embodiments, rAAV particles comprise the capsid of Anc80 or Anc80L65, as described in Zinn etal., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, tire rAAV particles comprise the capsid known as AAV.7m8 or AAV2.7m8 having amino acid insertions, as described in United States Patent Nos. 9, 193,956; 9458517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV.7m8, as described in United States Patent Nos. 9,193,956;

9,458,517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,585,971, such as AAVPHP.B. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al.. 2016, Gene Therapy 23: 857-862 and Georgiadis el al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsids of AAVUK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628.966; US 8,927,514; US 9.923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3. HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10 , HSC11, HSC12, HSC13, HSC 14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[00261] In some embodiments, rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524.446; 8,999,678; 8,628,966; 8,927.514; 8,734,809; US 9.284,357; 9,409.953; 9,169,299; 9,193,956; 9458517; and 9.587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more 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, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7.282.199; 7,906.111; 8,524,446; 8.999,678; 8,628.966; 8,927,514; 8.734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799;

PCT/EP2015/053335.

[00262] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g.. SEQ ID NO: 2), WO 2005/033321 (see, e.g.. SEQ ID NOs: 123 and 88), WO 03/042397 (see, e.g.. SEQ ID NOs: 2, 81, 85, and 97), WO 2006/068888 (see, e.g.. SEQ ID NOs: 1 and 3-6), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38) W02009/104964 (sec, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31), W0 2010/127097 (sec, e.g., SEQ ID NOs: 5-38), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more 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, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (sec, e.g., SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03/042397 (see, e.g.. SEQ ID NOs: 2, 81, 85, and 97), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31), W0 2010/127097 (see, e.g., SEQ ID NOs: 5-38), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5- 10).

[00263] Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282.199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803: 2015/0126588: 2017/0067908: 2013/0224836: 2016/0215024: 2017/0051257; International Patent Application Nos. PCT/US2015/034799; PCI7EP2015/053335; WO 2003/052051, WO 2005/033321, WO 03/042397, WO 2006/068888, WO 2006/110689, W02009/104964, W0 2010/127097, and WO 2015/191508, and U.S. Appl. Publ. No. 20150023924.

[00264] The provided methods are suitable for use in the production of recombinant AAV encoding a transgene. In certain embodiments, the transgene is from Tables 2A-2C. In some embodiments, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted tenninal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter/enhancers, b) a polyA signal, and c) optionally an intron; and (3) nucleic acid sequences coding for a transgene. In other embodiments for expressing an intact or substantially intact monoclonal antibody (mAb), the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter/enhancers, b) a polyA signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally a heavy chain Fc region. In still other embodiments for expressing an intact or substantially intact mAb, the rAAV genome comprises a vector comprising the following components: ( 1) AAV inverted terminal repeats that flank an expression cassette: (2) regulatory control elements, such as a) promoter/enhancers, b) a polyA signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the heavy chain Fab of an anti-VEGF (c.g., scvacizumab, ranibizumab, bcvacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651, ), anti-ALKl (e.g., ascrinvacumab), anti-C5 (e.g., tesidolumab and eculizumab). anti-CD105 (e.g.. carotuximab), anti-CClQ (e.g.. ANX-007), anti- TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), anti-IE6R (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 mAb (e.g., lanadelumab). 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., lampalizumab), or anti-MMP9 (e.g., andecaliximab); optionally an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, such as an IgG isotype amino acid sequence IgGl, IgG2 or IgG4 or modified Fc thereof; and the light chain of an anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab. and brolucizumab), anti-EpoR (e.g., LKA-651, ), anti-ALKl (e.g., ascrinvacumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g.. carotuximab), anti-CClQ (e.g.. ANX-007). anti- TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), 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 mAb (e.g., lanadelumab). anti-lTGA4 (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., lampalizumab), or anti-MMP9 (e.g., andecaliximab); wherein the heavy chain (Fab and optionally Fc region) and the light chain are separated by a self-cleaving furin (F)/F2A or flexible linker, ensuring expression of equal amounts of the heavy and the light chain polypeptides.

Table 2A

Table 2B

Table 2C

[00265] In some embodiments, the rAAV particles are rAAV viral vectors encoding an anti- VEGF Fab. In specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding an anti-VEGF Fab. In more specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding ranibizumab. In some embodiments, tire rAAV particles are rAAV viral vectors encoding iduronidase (IDUA). In specific 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 specific embodiments, the rAAV particles are rAAV9- based viral vectors encoding IDS. In some embodiments, the rAAV particles are rAAV viral vectors encoding a low-density lipoprotein receptor (LDLR). In specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding LDLR. In some embodiments, the rAAV particles are rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In specific embodiments, the rAAV particles are rAAV9-based viral vectors encoding TPP1. In some embodiments, the rAAV particles are rAAV viral vectors encoding non-membrane associated splice variant of VEGF receptor 1 (sFlt-1). In some embodiments, the rAAV particles are rAAV viral vectors encoding gamma-sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated 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), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid 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 (AP0A2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alphaglucosidase (GAA), alpha-galactosidase (GLA). beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1 -antitrypsin (AAT). phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN 1). survival motor neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrially encoded NADFLubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysfcrlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgGl) Fc fusion. In some embodiments, the gene product is a dystrophin or a microdystrophin. In some embodiments, the gene product is a microRNA, snRNA or antisense RNA.

[00266] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids arc rAAV2/8 or rAAV2/9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g.. Duan et l.. 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).

[00267] In additional embodiments, rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid protein is a chimeric of 2 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.rhlO, 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. [00268] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In certain embodiments, a self-complementary vector, e g., scAAV, can be used (see, e.g.. Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al. 2001, Gene Therapy. Vol. 8. Number 16. Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[00269] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

[00270] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 or AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more 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, to the VP1, VP2 and/or VP3 sequence of AAV8 capsid protein.

[00271] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has an AAV9 capsid protein at least 80% or more 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, to the VP1, VP2 and/or VP3 sequence of AAV9 capsid protein.

[00272] In additional embodiments, the rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, the rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2. AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, 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 comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrli.10, AAVhu.37, AAVrh.20, and AAVrh.74.

[00273] In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. AAV14, AAV15 and AAV16, AAV.rh8. AAV.rhlO. 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 additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype 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 comprise a pseudotyped rAAV particle containing AAV8 capsid protein. In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle comprised of AAV9 capsid protein. In some embodiments, tire pseudotyped rAAV8 or rAAV9 particles arc rAAV2/8 or rAAV2/9 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., 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). [00274] In additional embodiments, the rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, 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 comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10. rAAVrhlO, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. AAV14, AAV15 and AAV16, AAV.rh8. AAV.rhlO. 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 comprise an AAV capsid protein chimeric of AAV9 capsid protein 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, AAVhu.37. AAVrh.20, and AAVrh.74.

METHODS FOR ISOLATING RAAV PARTICLES

[00275] In some embodiments, the disclosure provides methods for producing recombinant adeno-associated vims (rAAV) particles, comprising isolating rAAV particles from a feed comprising an impurity (for example, rAAV production culture). In some embodiments, a method for producing recombinant adeno-associated vims (rAAV) particles described herein comprises (a) isolating rAAV particles from a feed comprising an impurity (for example, rAAV production culture), and (b) formulating the isolated rAAV particles to produce the formulation.

[00276] In some embodiments, the disclosure further provides methods for producing a pharmaceutical unit dosage of a formulation comprising isolated recombinant adeno-associated vims (rAAV) particles, comprising isolating rAAV particles from a feed comprising an impurity (for example, rAAV production culture), and formulating the isolated rAAV particles. [00277] Isolated rAAV particles can be isolated using methods known in the art. In some embodiments, methods of isolating rAAV particles comprises downstream processing such as, for example, harvest of a cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination(s) thereof. In some embodiments, downstream processing includes at least 2, at least 3, at least 4, at least 5 or at least 6 of: harvest of a cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration. In some embodiments, downstream processing comprises harvest of a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises clarification of a harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises clarification of a harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, downstream processing docs not include centrifugation. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV9 serotype.

[00278] In some embodiments, a method of isolating rAAV particles produced according to a method described herein comprises harvest of a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith 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 of isolating rAAV particles described herein comprises harvest of a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles produced according to a method described herein comprises clarification of a harvested cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith 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 of isolating rAAV particles described herein comprises clarification of a harvested cell culture, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles produced according to a method described herein comprises clarification of a harvested cell culture by depth filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith 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 of isolating rAAV particles described herein comprises clarification of a harvested cell culture by depth filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method docs not include centrifugation. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV9 serotype.

[00279] Numerous methods are known in the art for production of rAAV particles, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus-AAV hybrids, herpesvirus-AAV hybrids and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles all require; (1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549. or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), mammalian cell lines such as Vero, or insect- derived cell lines such as SF-9 in the case of baculovirus production systems; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production. In some embodiments, the suitable helper virus function is provided by a recombinant polynucleotide described herein or a plasmid described herein. Suitable media known in the art may be used for the production of rAAV vectors. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety.

[00280] rAAV production cultures can routinely be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, rAAV production cultures include attachmentdependent cultures which can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures may also include 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-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, Pcr.C6 cells, chicken embry o cells or SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system. 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 are known in the art for production of rAAV particles, including for example, the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No. 20120122155, each of which is incorporated herein by reference in its entirety.

[00281] In some embodiments, the rAAV production culture comprises a high density cell culture. In some embodiments, the culture has a total cell density of between about lxl0E+06 cells/ml and about 30xl0E+06 cells/ml. In some embodiments, more than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293 derived cells (e.g., EIEK293T 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.

[00282] In additional embodiments of the provided method the rAAV production culture comprises a suspension culture comprising rAAV particles. Numerous suspension cultures are known in the art for production of rAAV particles, including for example, the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No. 20120122155, each of which is incorporated herein by reference in its entirety. In some embodiments, the suspension culture comprises a culture of mammalian cells or insect cells. In some embodiments, the suspension culture comprises a culture 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 comprises a culture of HEK293 cells.

[00283] In some embodiments, methods for the production of rAAV particles encompasses providing a cell culture comprising a cell capable of producing rAAV adding to the cell culture a histone deacetylase (HD AC) inhibitor to a final concentration between about 0.1 mM and about 20 mM; and maintaining the cell culture under conditions that allows production of the rAAV particles. In some embodiments, the HDAC inhibitor comprises a short-chain fatty acid or salt thereof. In some embodiments, the HDAC inhibitor comprises butyrate (e.g., sodium butyrate), valproate (e.g., sodium valproate), propionate (e.g., sodium propionate), or a combination thereof. [00284] In some embodiments, rAAV particles are produced as disclosed in WO 2020/033842, which is incorporated herein by reference in its entirety.

[00285] Recombinant AAV particles can be harvested from rAAV production cultures by harvest of the production culture comprising host cells or by harvest of the spent media from the production culture, provided tire cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact host cells. Recombinant AAV particles can also be harvested from rAAV production cultures by lysis of the host cells of the production culture. Suitable methods of lysing cells are also known in the art and include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.

[00286] At harvest, rAAV production cultures can contain one or more of the following: (1) host cell proteins; (2) host cell DNA: (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components including, for example, serum proteins, amino acids, transferrins and other low molecular weight proteins. rAAV production cultures can further contain product-related impurities, for example, inactive vector forms, empty viral capsids, aggregated viral particles or capsids, mis-folded viral capsids, degraded viral particle.

[00287] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 mm or greater pore size known in the art. In some embodiments, clarification of tire harvested cell culture comprises sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the production culture harvest does not include centrifugation.

[00288] In some embodiments, harvested cell culture is clarified using filtration. In some embodiments, clarification of the harvested cell culture comprises depth filtration. In some embodiments, clarification of the harvested cell culture further comprises depth filtration and sterile filtration. In some embodiments, harvested cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises a depth filtration media. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train comprises two depth filtration media. In some embodiments, the filter train comprises a sterile filtration media. In some embodiments, the filter train comprises 2 depth filtration media and a sterile filtration media. In some embodiments, the depth filter media is a porous depth filter. In some embodiments, the filter train comprises Clarisolve® 20MS. Millistak+® C0HC, and a sterilizing grade filter media. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® C0HC. and Sartopore® 2 XLG 0.2 pm. In some embodiments, the harvested cell culture is pretreated before contacting it with the depth filter. In some embodiments, the pretreating comprises adding a salt to the harvested cell culture. In some embodiments, the pretreating comprises adding a chemical flocculent to the harvested cell culture. In some embodiments, the harvested cell culture is not pre-treated before contacting it with the depth filter.

[00289] In some embodiments, the production culture harvest is clarified by filtration are disclosed in WO 2019/212921, which is incorporated herein by reference in its entirety. [00290] In some embodiments, the rAAV production culture harvest is treated with a nuclease (e.g., Benzonase®) or endonuclease (e g., endonuclease from Serratia marcescens) to digest high molecular weight DNA present in the production culture. The nuclease or endonuclease digestion can routinely be performed under standard conditions known in the art. For example, nuclease digestion is performed at a final concentration of 1-2.5 units/ml of Benzonase® at a temperature ranging from ambient to 37°C for a period of 30 minutes to several hours.

[00291] Sterile filtration encompasses filtration using a sterilizing grade filter media. In some embodiments, the sterilizing grade filter media is a 0.2 or 0.22 pm pore filter. In some embodiments, the sterilizing grade filter media comprises polyethersulfone (PES). In some embodiments, the sterilizing grade filter media comprises polyvinylidcnc fluoride (PVDF). In some embodiments, the sterilizing grade filter media has a hydrophilic heterogeneous double layer design. In some embodiments, the sterilizing grade filter media has a hydrophilic heterogeneous double layer design of a 0.8 pm pre-filter and 0.2 pm final filter membrane. In some embodiments, the sterilizing grade filter media has a hydrophilic heterogeneous double layer design of a 1.2 pm pre-filter and 0.2 pm final filter membrane. In some embodiments, the sterilizing grade filter media is a 0.2 or 0.22 pm pore filter. In further embodiments, the sterilizing grade filter media is a 0.2 pm pore filter. In some embodiments, the sterilizing grade filter media is a Sartopore® 2 XLG 0.2 pm, Durapore™ PVDF Membranes 0.45pm, or Sartoguard® PES 1.2 pm + 0.2 pm nominal pore size combination. In some embodiments, the sterilizing grade filter media is a Sartopore® 2 XLG 0.2 pm.

[00292] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatographic medium, for example, affinity chromatography medium. Large scale concentration of viruses using TFF ultrafiltration has been described by Paul et al., Human Gene Therapy 4:609-615 (1993). TFF concentration of the clarified feed enables a technically manageable volume of clarified feed to be subjected to chromatography and allows for more reasonable sizing of columns without the need for lengthy recirculation times. In some embodiments, the clarified feed is concentrated between at least twofold and at least ten-fold. In some embodiments, the clarified feed is concentrated between at least ten-fold and at least twenty-fold. In some embodiments, the clarified feed is concentrated between at least twenty-fold and at least fifty-fold. In some embodiments, the clarified feed is concentrated about twenty-fold. One of ordinary skill in the art will also recognize that TFF can also be used to remove small molecule impurities (e.g., cell culture contaminants comprising media components, serum albumin, or other serum proteins) form the clarified feed via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration comprises the use of between about 3 and about 10 diafiltration volume of buffer. In some embodiments, the diafiltration comprises the use of about 5 diafiltration volume of buffer. One of ordinary skill in the art will also recognize that TFF can also be used at any step in the purification process where it is desirable to exchange buffers before performing the next step in the purification process. In some embodiments, the methods for isolating rAAV from the clarified feed described herein comprise the use of TFF to exchange buffers.

[00293] Affinity chromatography can be used to isolate rAAV particles from a composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from the clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from the clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art and include without limitation, AVB Sepharose™, POROS™ Capture Select™ AAVX affinity resin, POROS™ CapturcSclcct™ AAV9 affinity’ resin, and POROS™ Capture Select™ 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™ AAV 8 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAVX affinity resin.

[00294] 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 polish step. Suitable anion exchange chromatography media are known in the art and include without limitation, UNOsphere™ Q (Biorad, Hercules, Calif), and N-charged amino or imino resins such as e.g., POROS™ 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI -based resins known in the art (U.S. Pat. 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 comprises a quaternary amine. In some embodiments, the anion exchange media is a monolith anion exchange chromatography resin. In some embodiments, the monolith anion exchange chromatography media comprises glycidylmethacrylate-ethylenedimethacrylate or styrene-divinylbenzene polymers. In some embodiments, the monolith anion exchange chromatography media is selected from the group consisting of CIMmultus™ QA-1 Advanced Composite Column (Quaternary amine), CIMmultus™ DEAE-1 Advanced Composite Column (Diethylamino), CIM® QA Disk (Quaternary amine), CIM® DEAE, and CIM® EDA Disk (Ethylene diamino). In some embodiments, the monolith anion exchange chromatography media is CIMmultus ™ QA-1 Advanced Composite Column (Quaternary amine). In some embodiments, the monolith anion exchange chromatography media is CIM®¹ QA Disk (Quaternary amine). In some embodiments, the anion exchange chromatography media is CIM QA (BIA Separations, Slovenia). In some embodiments, the anion exchange chromatography media is BIA CIM® QA- 80 (Column volume is 80mL). One of ordinary skill in the art can appreciate that wash buffers of suitable ionic strength can be identified such that the rAAV remains bound to the resin while impurities, including without limitation impurities which may be introduced by upstream purification steps are stripped away.

[00295] In some embodiments, anion exchange chromatography is performed according to a method disclosed in WO 2019/241535, which is incorporated herein by reference in its entirety. [00296] In some embodiments, a method of isolating rAAV particles comprises determining the vector genome titer, capsid titer, and/or the ratio of full to empty capsids in a composition comprising the isolated rAAV particles. In some embodiments, the vector genome titer is determined by quantitative PCR (qPCR) or digital PCR (dPCR) or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is determined by serotype-specific ELISA. In some embodiments, the ratio of full to empty capsids is determined by Analytical Ultracentrifugation (AUC) or Transmission Electron Microscopy (TEM).

[00297] In some embodiments, the vector genome titer, capsid titer, and/or the ratio of full to empty capsids is determined by spectrophotometry, for example, by measuring the absorbance of the composition at 260 nm; and measuring the absorbance of the composition at 280 nm. In some embodiments, the rAAV particles are not denatured prior to measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured prior to measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using a spectrophotometer. In some embodiments, tire absorbance of the composition at 260 nm and 280 nm is detennined using an HPLC. In some embodiments, the absorbance is peak absorbance. Several methods for measuring the absorbance of a composition at 260 nm and 280 nm are known in the art. Methods of determining vector genome titer and capsid titer of a composition comprising the isolated recombinant rAAV particles are disclosed in WO 2019/212922, which is incorporated herein by reference in its entirety.

[00298] In additional embodiments the disclosure provides compositions comprising isolated rAAV particles produced according to a method described herein. In some embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. [00299] As used herein the term "pharmaceutically acceptable" means a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is 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, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example in administering rAAV isolated according to the disclosed methods to a subject. Such compositions include solvents (aqueous or nonaqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Pharmaceutical compositions and delivery systems appropriate for rAAV particles and methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed.. Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Phannaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins. Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[00300] In some embodiments, the composition is a pharmaceutical unit dose. A "unit dose” refers to a physically discrete unit suited as a unitary dosage for the subject to be treated; each unit containing a predetermined quantity optionally in association with a phannaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dose forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or deli very in vivo. Individual unit dose fonns can be included in multi-dose kits or containers. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, and recombinant virus particles, and pharmaceutical compositions thereof can be packaged in single or multiple unit dose form for ease of administration and uniformity of dosage. In some embodiments, the composition comprises rAAV particles comprising an AAV capsid protein from an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16. AAV.rh8, AAV.rhlO, AAV.rh20, AAV.H139, 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 AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9.

EXAMPLES

EXAMPLE 1. MOLECULAR DESIGN OF CONTROLLABLE RECOMBINANT AAV EXPRESSION SYSTEMS FOR ENHANCED VECTOR PRODUCTION.

[00301] Recombinant adeno-associated virus (rAAV) is the leading tool for gene therapy applications. Despite the evidence of remarkable clinical efficacy, low production yields remain a major challenge in manufacturing of rAAV vectors. Key aspects of vector production include the molecular design of rAAV expression plasmids. Here, we present the design of an improved, controllable Rep/Cap expression system utilizing a truncated p5 promoter and a p5 cis-regulatory element at the 3 ’ end in combination with (i) a heterologous promoter to drive Cap expression, and (ii) an additional copy of the rep52/40 gene to overexpress short Rep proteins. Demonstrated herein is that Rep78 is essential for efficient rAAV8 production in HEK293 cells, and a higher ratio of short Rep to long Rep proteins enhances genome packaging. Through Helper sequence deletion variants, regulators and open reading frames (ORFs) within the Helper plasmid were identified that contribute to high-yielding rAAV8 production. While E2A intron and L4-33k/22k are integral to optimal production, the use of E4orf6 and 6/7 subset enhances rAAV8 titer by 85%. This study demonstrates that configuring and controlling the expression of the different AAV genetic elements contribute toward high productivity and product quality (fiill/empty capsid ratio).

[00302] Recombinant adeno-associated viruses (rAAV) are a growing part of biopharmaceutical development pipelines. Despite the evidence of outstanding clinical efficacy, the high cost of manufacture and vector dosages required has limited the economic viability of AAV-mediated gene therapies. [Reference 1,2] The three-plasmid transient transfection in HEK293 cells is a widely utilized method for producing rAAV. One of the main challenges in creating high-yielding AAV expression systems and generating AAV packaging cell lines is the ElA-mediated transactivation of promoters p5 and pl 9. [Reference 3] The former promoter controls the gene expression of AAV replication proteins Rcp78/68 that arc known to be cytostatic or cytotoxic [Reference 4,5] but are also required for transactivation of promoters pl9 (transcribing Rep52/40) and p40 (transcribing capsid (Cap)). [Reference 6] On the other hand, overexpression of Cap has been shown to be advantageous for the optimal production of rAAV. [Reference 7,8] To this end, Rep78/68 (large Rep), Rep52/40 (small Rep), and Cap expression have to be regulated independently. Furthermore, Rep78 represses adenovirus Helper promoters E1A, E2A and E4 and therefore needs to be controlled tightly during rAAV production and cell growth. [Reference 9] Engineering AAV expression vectors for improved manufacturability in mammalian cell factories remains a highly desirable objective.

[00303] In its natural context, AAVs achieve a precise expression stoichiometry of multiple genes within a compact genome (4.7 kb) using a combination of internal (within open-reading frame (ORFs)) promoters, overlapping ORFs, differential mRNA splicing, alternative translation start sites (with varying initiation rates) and feedback loops (using transactivators or repressors). [Reference 3] Various vector engineering strategies have been utilized to improve rAAV expression in cell hosts. Examples include use of inducible promoters, [Reference 4] intron insertion, [Reference 10] Kozak/start codon mutations [Reference 11,12] and a four-plasmid system [Reference 13] to modulate the rep and cap gene expression, as well as a hybrid Rep to improve genome packaging efficiency. [Reference 14] Further efforts to boost rAAV yields targeted the Helper plasmid via utilization of human bocavirus 1 Helper genes [Reference 15] or design-of-experiment (DoE) approach to optimize the Helper, packaging and transgene plasmid ratios. [Reference 16] Despite these improvements, there remain limited reports on the impact of individual (sub)components that need to be considered when designing an AAV vector and how they can be controlled and enhanced.

[00304] In this study, components and regulators of rAAV8 transient expression in HEK293 cells were identified by mechanistically dissecting the packaging and Helper plasmids. The impact of p5 cis-rcgulatory clement, endogenous and heterologous promoters, introns, removal of ORFs and up/downregulation of specific genes on rAAV product titer were systematically determined. Using optimized split Rep/Cap and Helper plasmids, further demonstrated was that it is possible to control genome titer and product quality (full/empty capsid ratio) in a transient rAAV expression system.

[00305] 2.1 Plasmid construction: Helper plasmid pAdDeltaF6 was constructed by Dr. James M. Wilson and colleagues at UPcnn (Addgcnc #112867) and then the AmpR gene was replaced with the KanR gene to create pAdDeltaF6-KanR. Hie Rep/Cap and Helper plasmid variants were constructed by PCR amplification (Q5 High-Fidelity 2x Master Mix; NEB), site-directed mutagenesis (Q5 Site-Directed Mutagenesis kit; NEB) and/or gene synthesis (Eurofins Genomics). PCR products were purified using QIAquick PCR Purification kit (Qiagen), gel extraction was performed using QIAquick Gel Extraction kit (Qiagen). Restriction enzymes were obtained from NEB. Ligation was performed using T4 DNA ligase (NEB), assembly of multiple DNA fragments was performed using NEBuilder HiFi DNA Assembly Master Mix (NEB). The sequence regions of the relevant promoters are detailed in Table 3. The sequence mutations are detailed in Table 4. Rep/Cap plasmids were amplified in DH5a competent cells (Thermo Fisher Scientific), Helper plasmids were amplified in NEB Stable competent cells (NEB). Clonally derived plasmids were purified using QIAprep Spin Miniprep kit (Qiagen) or QIAGEN Plasmid Plus kit (Qiagen). Tire sequence of all plasmid constructs was confirmed by restriction enzyme analysis and DNA sequencing (Eurofms Genomics).

Table 3. Sequences of AAV endogenous promoters and the CMV promoter. Nucleotide numbers refer to NCBI GenBank.

Table 4. Example sequence mutations for inactivation or optimization of Rep/Cap elements.

[00306] 2.2 HEK293 cultures: Suspension-adapted HEK293 cells were cultured in Dynainis medium (Thermo Fisher Scientific) supplemented with L-glutamine (Thermo Fisher Scientific). Cells were maintained in Erlenmeyer flasks (Coming) at 37°C, 140 rpm under 5% CO2, 85% humidity and were sub-culturcd every 3-4 days by seeding at 3* 105 viable cclls/mL. Cell viability and viable cell density (VCD) were measured using a Vi-CELL XR (Beckman Coulter). [00307] 2.3 PEI-mediated transient vector production: rAAV8 production was performed by triple transfection and in shallow-well 24-well plates (Coming) using the Deutz system as previously described. [Reference 17] Briefly, cells were sub-cultured in an Erlenmeyer flask and grown to 4x 106 cells/mL. Prior to transfection, aliquots of 700 pL were added to each well of 24- wcll plate. Plasmid DNA (weight ratio of 1 :2:0.1 for packaging plasmid, Helper plasmid, transgene plasmid) [Reference 60] and PEIpro (Polyplus-transfection) were each pre-diluted in Dynamis medium, combined and incubated at room temperature for 10 min before being added into culture. Transfected cells were cultured for 72 h at 37°C, 230 rpm under 5% CO2, 85% humidity.

[00308] 2.4 Quantification of viral genome titer by ddPCR: Intra and extracellular rAAV8 titer was quantified by digital droplet PCR (ddPCR) as previously described. [Reference 17] Briefly, 10x cell lysis buffer containing 1 x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) was added to cell culture and incubated at 37°C for 1 h. Samples were centrifuged to remove cell debris and the supernatant was treated with DNase I (Roche), followed by dilution in GeneAmp PCR Buffer I (Thermo Fisher Scientific) containing 0.02% UltraPure Salmon Sperm DNA Solution (Thermo Fisher Scientific) and 0.1% Pluronic F-68 Non-ionic Surfactant (Thermo Fisher Scientific). Viral genome (VG) titer was quantified using QX200 Droplet Digital PCR system (Bio-Rad) and primers and a probe targeting the poly A sequence of the transgene plasmid harboring a CAG promoter and a GFP gene. The absolute VG titer was determined using the Quantasoft analysis software (Bio-Rad).

[00309] 2.5 Quantification of intact capsid titer: Total capsid titer quantification was performed using the AAV8 titration ELISA (Progen) according to the manufacturer’s instructions. Total cell lysis supernatant diluted in 1 x AS SB assay buffer (Progen). OD values at 450 nm and 650 nm (background absorbance) were measured using a SpectraMax iD5 microplate reader (Molecular Devices).

[00310] 2.6 Statistics: Microsoft Excel 2016 (Microsoft) was used to analyze the difference between the means (normalized titers or intact capsids) of a plasmid construct and the control. Analysis was performed using unpaired Student’s t-test with /?-value < 0.05 was considered significant.

[00311] 3.1 A heterologous promoter and inclusion of Cap intron enable a controllable

Rep/Cap plasmid system: Previous studies showed that unregulated overexpression of Rep78/68 inhibited rAAV production, while reduced level of Rep78/68 enhanced rAAV titers. [Reference 18,19,20] Further, promoter p5 acts a cis-regulatory element where its deletion was shown to cause downregulation of promoters pl9 and p40. [Reference 6,21.22] The standard Rep/Cap plasmid comprised two modifications: (i) truncation of the p5 promoter to attenuate expression of the large Rep proteins, and (ii) introduction of a p5 promoter downstream of the AAV cap region to retain the expression of pl9 and p40 (Figure 1A). [Reference 61] As shown in Figure IB, tire improved Rep/Cap plasmid (Rep/Cap 1.2; Control) displayed a 32% increase in rAAV8 titer over the conventional packaging plasmid (p < 0.01), reaching 10¹⁴ VG/L in serum-free media. Triple transfection was performed at a weight ratio of 1 :2:0.1 (packaging plasmid. Helper plasmid, transgene plasmid). [Reference 60] While it is relatively easy to regulate rep78/68, control of rep52/40 and cap expression would involve the complex multimeric gene assembly in which the pl9 and p40 promoters are located within the rep coding sequences.

[00312] In order to enable control of cap gene expression, the rep and cap genes were split by cloning the p40 promoter and cap open reading frame (ORF) downstream of the rep gene stop codons. To prevent expression of truncated viral gene products, the TATA box and Initiator (Inr) of the internal p40 promoter as well as the start codon of the internal cap remnant were inactivated by mutations while preserving the functionality of the Rep proteins (i.e. without altering the encoded amino acid; Table 4). Measurement of rAAV8 titer at 72 h post-transfection is shown in Figure ID. Separation of tire rep and cap genes, either without or with a downstream p5 promoter (Rep/Cap 2.0 and 2. 1, respectively) dramatically reduced the rAAV titer to <15% compared to the control. Addition of a poly A for the rep ORFs (Rep/Cap 2.2) for independent termination of transcription did not result in noticeable increase in rAAV titer. While inclusion of cap intron (Rep/Cap 2.3) for efficient post-transcriptional processes increased the titer by two- fold compared to Rep/Cap 2. 1 (indicative of a critical element), it was only one-third of the control titer. Without being bound by a specific theory', it was surmised that the transcription factor (TF) binding sites within the upstream (inactivated) p40 promoter acted as competing binding elements [Reference 23] resulting in reduced cap transcriptional activity.

[00313] In order to evaluate whether the upstream p40 promoter corresponded to TF decoy sites", the downstream p40 was replaced with a heterologous promoter, the human CMV. [Reference 24] As shown in Figure ID, employing the CMV promoter to drive cap expression without its intron (Rep/Cap 3.0-3.3) resulted in slight increases in rAAV titer compared to the Rep/Cap 2.0- 2.2 constructs, with Rep/Cap 3.3 exhibiting 25% of the control titer. The inclusion of the cap intron (Rep/Cap 3.4) significantly increased the rAAV production to 70% of the control titer, and the addition of a p5 promoter downstream (Rep/Cap 3.5) restored the titer to the control level — consistent with the enhancer function associated with p5. [Reference 21,22] While the addition of a second copy of p5 cis-regulatory element directly upstream of the CMV promoter (Rep/Cap 3.6) did not increase the titer relative to the Rep/Cap control, introducing a poly A downstream of the rep gene (Rep/Cap 3.7) further enhanced the rAAV yield to 116% (albeit statistically insignificant, p = 0.075). Without being bound by a specific theory , the observations made above are consistent with an understanding that (i) a heterologous promoter is required to drive efficient transcription of the split cap gene, (ii) the cap intron is a key regulator of Cap expression, and (iii) a poly A can be used to enable independent control of the rep genes for improved rAAV production. Tire split Rep/Cap system will permit attunement of the expression level of both the Rep proteins and the capsids to increase production of rAAV for use in gene therapy, for example through modification of promoters and Kozak sequences, rearrangement of the genes, as well as codon (de)optimization.

[00314] 3.2 Rep78 protein is essential while higher ratio of Rep52/40 to Rep78/68 proteins enhances rAAV8 production: Previous studies suggested that the functions between Rep78 and Rep68, and between Rep52 and Rep40 were redundant. [Reference 25,26] Further, Rep52/40 proteins (in contrast to Rep78/68) were found not to inhibit the grow th of primary, transfonned and immortalized cells. [Reference 27] Therefore, it was hypothesized that rAAV production can be increased via (i) complete ablation of highly cytotoxic replication protein Rep78. and (ii) overexpression of Rep52/40 to enhance the packaging and accumulation of single-stranded viral genome [Reference 28] without inducing cytotoxicity. With regard to the latter, the constraint in regulating p 19 is due to the position of this promoter which is located within the protein coding sequence of Rep78/68. To illustrate this, the weak Kozak sequence of Rep/Cap 3.5 (TACATGG. start codon underlined) [Reference 29] was mutated to promote the short Rep expression (see Figure 7). Measurement of rAAV8 titer after transient transfection of HEK293 cells showed that mutating the TAC (tyrosine) to ATC (isolucine) within the Kozak diminished the rAAV production by -1000-fold (Rep/Cap 4.0; Figure 2) — implying a loss in Rep78/68 functionality, or uncontrolled overexpression of Rep52/40.

[00315] To test the hypothesis that rAAV8 can be produced in HEK293 cells that have one protein out of Rep78/68 and one protein out of Rep52/40, the Rep/Cap 3.5 plasmid (Figure 1C) was modified with deleted alternate splice site within the rep codons to produce only Rep68 and Rep40 proteins (Rep/Cap 4.1; Figure 2A). [Reference 13] As shown in Figure 2B, removal of rep78 and rep52 markedly reduced rAAV titer by 73% compared to the Rep/Cap 1.2 control. To determine whether the absence of rep52 was responsible for the titer reduction, a rep52/40 gene was introduced downstream of cap (Rep/Cap 4.2). To further overexpress the short Rep proteins, a second copy of p5 (cis-regulatory element for the p 19 promoter) was added upstream of the CMV (Rep/Cap 4.3), or substituted the pl9 with p5 promoter and mutated the weak Kozak sequence (Rep/Cap 4.4). Note that the p5 promoter is approximately twice as active as the pl9 promoter (Figure 8). The results showed that neither reintroduction of rep52 nor attempts to augment Rep52/40 expression resulted in noticeable improvements in rAAV titer compared to Rep/Cap 4.1.

[00316] Based on the above observations, a library of Rep/Cap plasmids was created with all four rep coding regions including an additional copy of rep52/40 downstream of cap to enhance the packaging and accumulation of single-stranded viral genome. The second copy of rep52/40 was driven by either promoter p 19 or p5 and with or without a mutated Kozak sequence (Rep/Cap 5.0-5.4; Figure 2A). As shown in Figure 2B, analysis of rAAV titer confirmed that p5 promoter at the 3 ' end was critical for maximal titers (see Rep/Cap 5.2 vs. 5.0). Importantly, the data demonstrated that the reintroduction of rep78 reinstated the rAAV production system with two constructs (Rep/Cap 5.3 and 5.4) significantly exceeded the Rep/Cap 1.2 control titer (-118%; p < 0.05). No further increase in rAAV titer was observed when a poly A was added between the long rep and cap genes (Rep/Cap 5.3 vs 5.4). In summary, even though either Rep78 or Rep68 alone may be sufficient for AAV DNA replication, [Reference 25] the results show that rAAV production in HEK293 cells is critically regulated by the full-length Rep78 — corroborating previous studies suggesting that Rep78 and Rep52 proteins are necessary for efficient viral production. [Reference 30,31] Without being bound by any theory', Rep78 (at a low level) is required for optimal rAAV DNA replication, and a higher ratio of short Rep (Rep52/40) to long Rep (Rep78/68) proteins may enhance genome packaging without inducing cytotoxicity in transient rAAV expression systems.

[00317] 3.3 E2A intron and L4-33k/22k proteins are required for optimal rAAV8 production: The pAdDeltaF6-KanR Helper plasmid (15.8 kb) utilized in this study was composed of the E2A (encoding DNA binding protein (DBP)), E4 and VA RNA regions derived from adenovirus-5 genome. Hie E2A is transcribed by two promoters, namely E2-early and E2- late, with DBP mRNA levels coming from the E2 -early promoter being dominant. Additionally, the E2-early promoter/intron sequence encodes the L4-33k/22k proteins on the opposite strand, driven by L4 promoter (Figure 3A). [Reference 32, 63] Even though the L4-22k/33k proteins have been indicated to play a role in adenovirus assembly, gene expression and viral DNA packaging, [Reference 33,34] their significance in rAAV production is largely undetermined. This represents a potential engineering target or elimination from the vector. With regard to the latter, minimizing plasmid size is desirable for enhanced transient production by increased transfection efficiency and copy numbers of required genes per DNA weight.

[00318] In order to evaluate the function of the E2A region, Helper plasmids were constructed containing a partially or fully deleted E2A intron (including a 77 bp exon contained within) [Reference 32, 63], with the former retaining the E2-latc promoter and L4-33k/22k coding sequence (E2Aminl and E2Amin2 Helper; Figure 3A). Triple transfection was performed as described above and rAAV8 titer was measured 72 h post-transfection. These data show that the E2Aminl and E2Amin2 reduced the titer to 82% and 37% of that deriving from E2A control, respectively (p < 0.001), suggesting a significant reduction in DBP sy nthesis. However, the L4- 33k/22k ORFs located in the E2A promoter/intron fragment did not allow tire conclusion that only the DBP contributed to the rAAV Helper function. To elucidate this, an L4 promoter-driven plasmid expressing only the L4-33k/22k proteins was constructed and co-transfected with the E2Amin2 Helper plasmid at equal molar ratio. This analysis demonstrated that the L4-33k/22k single gene co-expression (E2Amin2 + L4-33k/22k) resulted in a significant increase in rAAV titer compared to E2Amin2 (53% increase, p < 0.05; Figure 3B). As L4-33k mutant virus has been shown to produce only empty adenoviral capsids, [Reference 34] it was hypothesized that the L4-33k-deficient rAAV production suffered from a defect in viral DNA packaging resulting in a lower titer. It was inferred that the E2A intron and L4-33k/22k proteins were integral components for optimal, high-yield rAAV8 production. Further, this finding of a rAAV replication stimulating element represents a potential engineering target, for example via protein overexpression to enhance rAAV production.

[00319] 3.4 Helper plasmid comprising E4 orf6 and 6/7 subset enhances rAAV8 production: The E4 gene of adenovirus encodes seven proteins, namely E4 orf (open reading frame) 1, 2, 3, 3/4, 4, 6 and 6/7, each with different functions including promoting viral gene expression and replication as well as modulation of TF activities. [Reference 35] Among these, only the E4orf6 protein was thought to contribute to rAAV production and solely employed in a number of Helper plasmid variants. [Reference 36.37,38] Despite the minimal observed effect of other E4orfs on adenovirus growth in cultured cells, [Reference 38,39] we hypothesized that rAAV production could be optimized by specific combinations of the E4orf proteins.

[00320] In order to specifically determine the functional contribution of different E4orfs, the E4 gene was dissected by constructing Helper plasmids containing different subsets of the orfs (Figure 4A). This set of six plasmids was then tested for their ability to mediate AAV8 vector production. Measurement of rAAV titer after triple transfection of HEK293 cells with each Helper variation is show n in Figure 4B. As anticipated, the E4orf6 was capable of producing rAAV help equivalent to a full-length E4 gene (111%; p = 0.0773). Moreover, the data show that removal of orfl and 2 (i.c. E4orf2-6/7 and E4orf3-6/7 subsets) increased the rAAV titer by 22% and 37% (p<0.05), respectively, compared to the E4 control. In this regard, it was conjectured that the deletion of these two redundant orfs (where their functions are largely undefined) [Reference 35] increased the abundances of other orf mRNAs spliced from the same precursor mRNA transcript. Deletion of orfl to 3 (E4orf4-6/7 subset) decreased the rAAV titer to the control level — this was not unexpected considering that orf3 (similar to orf6) functions in promoting viral gene expression and replication. [Reference 38] Importantly, the data described herein shows that further deletion of orf4 while retaining orf6/7 (E4orf6-6/7 subset) significantly enhanced the titer to 163% of the E4 control titer (p < 0.001). This result is in line with the previous studies that identified orf4 as a negative regulator of E1A and E4 transcription [Reference 40] while orf6/7 modulates the activity' of the cellular transcription factor E2F. [Reference 42] The effect of E4orf3 on rAAV production was further examined by measuring the rAAV titer produced using a helper comprising the E4orf3 and orf6-6/7 subset (Figure 4C). Measurement of rAAV titer after triple transfection of HEK293 cells with each Helper variation (Figure 4C) was performed essentially as described in 3.3 hereinabove. The data shows that including of E4orf3 and addition to E4orf6-6/7 significantly enhanced the titer relative to both the E4 control and relative to the helper comprising E4orf6-6/7. These results show that rAAV production in HEK293 cells can be enhanced via removal of redundant E4orfs.

[00321] 3.5 Engineered Rep/Cap and Helper plasmids can be used together to control rAAV8 gene expression: To evaluate whether the controllable Rep/Cap system could complement the engineered Helper to enable efficient rAAV production, E4orf6-6/7 Helper plasmid was utilized in combination with either Rep/Cap 3.7 or 5.4 plasmid. Additionally, previous studies showed that regulatory loops exist in which El A. DBP and E4orf6/7 proteins positively or negatively regulate promoters p5. El A, E2-early and E4 as well as transcription factor E2F, among others. [Reference 41,42] Therefore, it was evaluated whether substitution of constitutively active CMV promoter sequences for the E2A and/or E4 regulator}’ sequences in the E4orf6-6/7 Helper plasmid (Figure 5A) have positive/negative effects on rAAV vector production.

[00322] Measurement of rAAV8 titer after triple transfection of HEK293 cells with different Helper and Rep/Cap variant combinations is shown in Figure 5B. Tire result showed that both Rep/Cap 3.7 and 5.4 were incompatible with Helper E4orf6-6/7 (Helper 2.0) where they displayed 42% and 26% reduction in rAAV titer, respectively, relative to the Rep/Cap 1.2 control (p < 0.01). Without being bound by a theory, it is postulated that the use of the strong, highly complex CMV promoter in Rep/Cap 3.7 and 5.4 plasmids titrated away the limited pool of available TF molecules from the E4 promoter resulting in E4orf6-6/7 downregulation. [Reference 23,24] The use of CMV promoter to drive E4orf6-6/7 transcription (Helper 2.1) restored the rAAV titer comparable to Helper 2.0 with Rep/Cap 1.2 (83-110%). In contrast, substituting the E2 -early promoter with the CMV promoter (Helper 2.2) led to reduced titers especially when used in conjunction with Rep/Cap 1.2 and 5.4. Further decreases in rAAV level were observed with Helper 2.3 plasmid that harbored the CMV promoter to drive both E4orf6-6/7 and E2A expression — in general agreement with tire view that competing TF binding sites, as well as the reports that constitutive, high level E2A expression is incompatible within cell lines stably producing DBP. [Reference 43,44,45] The CMV promoter is about three times more active than the E2-early promoter (Figure 8).

[00323] Tire data in Figure 5B also show that Rep/Cap 5.4 with Helper 2.0 or 2.1 yielded higher titers compared to Rep/Cap 3.7. To expound this observation, a sub-panel of different Rep/Cap and Helper combinations was selected from Figure 5B as well as Figures ID and 2B (utilizing pAdDeltaF6-KanR plasmid: Helper 1.0), and quantified fully assembled, intact capsids to determine the ratio of full to empty particles. This analysis indicated that the use of the CMV promoter to drive Cap expression (Rep/Cap 3.7 and 5.4) with either Helper 1.0, 2.0 or 2.1, boosted total capsids by an average of ~3-fold compared to Rep/Cap 1.2 (Figure 6A), resulting in full/empty capsid ratio of < 9% (Figure 6B). Importantly, the analysis also revealed that all transfections utilizing Rep/Cap 5.4 (overexpressing Rep52/40 proteins) yielded relatively higher full to empty capsid ratios compared to Rep/Cap 3.7, indicating a higher rate of packaging and accumulation of single-stranded DNA progeny genomes (see also the accompanied increase in VG titer for Helper 2.0 and 2.1; Figure 5B). Very high full/empty ratios (up to 66%) were achieved using Helper 2.2 and 2.3 although this was largely due to considerable reductions in intact capsid abundance compared to other Helper variants. Taken together, these data demonstrate that it is possible to control both genome and total viral particle titer in a transient rAAV expression system. Tire novel library of CMV promoter sequences with variable strengths [Reference 24] is anticipated to enable systematic determination of the optimal Cap expression for maximal rAAV product titer and quality.

[00324] This study was designed to characterize the diverse components (ORFs) and regulators (promoters, introns) of the AAV transient triple transfection plasmid system underpinning the biomanufacturing processes, for example, of how the abundance (or absence) of the four Rep proteins affects the efficiency of rAAV production yield. Specifically, the data described herein indicates a sub-optimal rAAV8 production state when rep78 was removed. This finding is in line with the previous reports in which Rep78 was shown to be more efficient than Rep68 in producing infectious Rep-negative AAV, [Reference 25] and hence indirectly favors the vector design strategy that omitted rep68 [Reference 46] rather than rep78 [Reference 13] to mitigate Rep toxicity effects. Furthermore, this study identified the L4-33k/22k gene as an integral rAAV component, corroborating a recent unpublished study that showed a >20-fold decrease in rep and cap DNA in HeLa cells transfected with the 33k/22k-targeting siRNA [Reference 47] — suggesting a cell engineering opportunity for increased rAAV production, for example via overexpression of these adenovirus Helper proteins.

[00325] Another key finding of the work described herein is that E4 orfl, 2 and 4 are functionally redundant within the AAV expression system, consistent with the existing notion that a single E4orf6 protein is needed to produce rAAV vectors in HEK293 cells. [Reference 36.37,38] Critically, the results described herein reveal that rAAV titer can be enhanced by specific combinations of the E4orf proteins particularly orf6 and 6/7, or orf3 in combination with ofr6 and 6/7. The removal of redundant orfs also likely improved the expression of other orfs due to reduced splice sites. Without being bound by a theory, the latter protein, E4orf6/7 modulates the activity of the E2 -early promoter by forming a direct complex with transcription factor E2F and stabilizing the DNA-bound form. [Reference 41] As the E4 promoter is inhibited by the E2A product. [Reference 42] a regulatory loop exists in which E4orf6/7 protein increases E2A transcription while DBP negatively regulates E4 transcription. From a mechanistic perspective, one can assume that the advantage of this temporal coordination is restricted DBP “toxic” effects [Reference 43,44,45] during the bioproduction process thus ensuring maximal productivity. Replacing the E2 -early promoter with a constitutive, highly active promoter such as the CMV would result in uncontrollable E2A gene expression and consequently rapid cellular accumulation of DBP. Nevertheless, the cell concentration/viability data described herein showed no differences between the endogenous E2-early and CMV promoter-driven E2A constructs (data not shown). One can speculate that the detrimental effects of high DBP level in HEK293 cells were via negative regulation of capsid expression rather than direct exertion of cytotoxicity on the host cells.

[00326] Even though the heterologous CMV promoter is beneficial for tire production of rAAV from the split packaging vector system, product quality analysis showed that most of the capsids generated from these vectors were empty and therefore were unable to provide therapeutic benefits. This remains the case even when a second short rep gene was introduced to enhance packaging and accumulation of single-stranded viral genome. Nevertheless, it may be possible to circumvent this drawback by using specific cis-regulatory modules within the CMV promoter architecture (i.e. specific strengths) as previously reported [Reference 24] for defined capsid expression levels. Moreover, promoter activity in a given cell host is governed by a system specific combination of interactions between the promoter’s constituent TF binding sites and the availability of endogenous TFs. [Reference 24] Accordingly, the use of the CMV promoter to drive cap gene expression likely resulted in titration of TFs away from the endogenous E4 promoter affecting the expression level of E4orf proteins (and consequently rAAV titer; Figure 5). Expectedly, further bioinfomiatic analysis of regulatory elements within these promoters indicated significant (active) TF binding site overlaps between them (Figure 9). In this regard, vectors utilizing synthetic promoters designed de novo using specific TF binding site building blocks [Reference 48,49] are likely to be the solution for predictable stoichiometries of different AAV vector components in transient as well as stable systems. [Reference 50]

EXAMPLE 2. STABLE CELL LINE DEVELOPMENT FOR RECOMBINANT AAV PRODUCTION.

[00327] The triple plasmid transfection system in HEK293 cells is well established and commonly used for clinical and commercial manufacturing. Figure 10. In this system, one plasmid, often referred to as the trans plasmid, carries Rep and Cap genes and encodes proteins for virus replication and capsid formation. A second plasmid, often referred to as the helper plasmid, encodes the essential adenovirus helper genes (E4, E2A, and viral associated (VA) RNAs), and a third plasmid, often referred to as the cis plasmid, contains an expression cassette flanked by two inverted terminal repeats (ITRs), which is incorporated into the rAAV as its genome. The additional helper genes El A and E1B are expressed endogenously by the HEK293 cells. Major challenges for stable rAAV producing cell lines include the toxicity of the AAV Rep and Cap polypeptides, and the difficulty of coordinating the expression of more than 10 polypeptides necessary for efficient recombinant AAV production. Figure 11.

Design of inducible plasmids for Rep/Cap expression

[00328] 8 different configurations of inducible trans plasmids expressing an AAV Rep polypeptide and an AAV Cap polypeptide were designed and tested in a transient transfection based assay. Figures 12 and 14.

[00329] iTrans#! through iTrans#4 plasmids were designed to separate sequences expressing the long Rep78/68 polypeptides from the sequences expressing the short Rep 52/40 polypeptides. iTrans# 1 comprises, in 5' to 3' direction, a first tetracycline inducible promoter operably linked to an AAV2 Rep78/68 coding region comprising mutations that inactivate the p!9 and p40 promoter without altering the Rep polypeptide sequence encoded, a CMV promoter operably linked to an AAV8 Cap coding region and a second tetracycline inducible promoter operably linked to an AAV2 Rep52/40 coding region comprising mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequence encoded. The iTrans#2 and iTrans#3 plasmids differ from iTrans#! in that they express only long and one short Rep polypeptide. iTrans#2 comprises, in 5' to 3' direction, a first tetracycline inducible promoter operably linked to an AAV2 Rep78 coding region comprising mutations that inactivate the p 19 and p40 promoter without altering the Rep polypeptide sequence encoded, a CMV promoter operably linked to an AAV8 Cap coding region and a second tetracycline inducible promoter operably linked to an AAV2 Rep52 coding region comprising mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequence encoded. iTrans#3 comprises, in 5' to 3' direction, a first tetracycline inducible promoter operably linked to an AAV2 Rep68 coding region comprising mutations that inactivate the pl9 and p40 promoter without altering the Rep polypeptide sequence encoded, a CMV promoter operably linked to an AAV8 Cap coding region and a second tetracycline inducible promoter operably linked to an AAV2 Rep52 coding region comprising mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequence encoded. iTrans#4 differs from iTrans#! in that it comprises an inducible promoter for the expression of the Cap polypeptides. iTrans#4 comprises, in 5' to 3' direction, a first tetracycline inducible promoter operably linked to an AAV2 Rep78/68 coding region comprising mutations that inactivate the pl9 and p40 promoter without altering the Rep polypeptide sequence encoded, a second tetracycline inducible promoter operably linked to an AAV8 Cap coding region and a third tetracycline inducible promoter operably linked to an AAV2 Rcp52/40 coding region comprising mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequence encoded.

[00330] iTrans#5 was designed to lower Rep polypeptide expression by the use of an internal ribosomal entry site (IRES). iTrans#5 comprises, in 5' to 3' direction, a tetracycline inducible promoter operably linked to a transcription unit comprising AAV8 Cap coding region, and IRES and a AAV2 Rep78/68/52/40 coding region comprising mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequences encoded. Variants of iTrans#5 can be made, for example, by making nucleotide modifications in the Rep sequence to inactivate the pl9 promoter.

[00331] iTrans#6 through iTrans#8 were designed to modulate Rep polypeptide expression by driving Rep78 and Rep52 expression using separate promoters, wherein the promoter controlling Rep52 expression is included in an engineered intron added to tire Rep coding region. iTrans#6 through iTrans#8 comprise, in 5' to 3' direction, a (1) first tetracycline inducible promoter operably linked to (2) a transcription unit comprising (i) an upstream Rep coding region, (ii) an intron comprising a second tetracycline inducible promoter and (iii) a downstream Rep coding region comprising a Rep52 coding sequence, wherein the second tetracycline promoter is operably linked to the downstream Rep coding region comprising a Rep52 coding sequence, and wherein the Rep coding regions comprise mutations that inactivate the p 19 and p40 promoters without altering the Rep polypeptide sequence encoded; and (3) a CMV promoter operably linked to an AAV8 Cap coding region. iTrans#6 through iTrans#6 use different intron sequences. iTrans#6, iTrans#7 and iTrans#8 comprise the introns #1 (SEQ ID NO: 57), #2 (SEQ ID NO: 58). and #3 (SEQ ID NO: 59), respectively, comprising the second tetracycline inducible promoter. [00332] To assess the ability of iTrans#! through iTrans#8 to support inducible rAAV production, a HEK293 derived cell was transiently transfected with the Trans plasmids in combination with a helper plasmid and a cis plasmid encoding a GFP-based reporter transgene. Tire HEK293 derived host cell expressed a tetracycline dependent transactivator capable of activating transcription by the tetracycline inducible promoters used in the iTrans#! through iTrans#8 constructs. Following transfection, the cells were incubated in the presence of 0. 100, 500 or 1,000 ng/ml Doxycycline. rAAV titer produced were measured using digital PCR. Tire results are shown in Figure 14. iTrans#5 and iTrans#7 were able to promote Doxycycline dependent rAAV production. All other trans plasmids tested produced a higher rAAV titer in the absence in Doxycycline than in the presence of Doxycycline at any concentration tested.

Design of inducible helper plasmids

[00333] An inducible helper plasmid was generated to determine whether it can be combined with the iTrans#! through iTrans#8 plasmids described herein for use in rAAV production. The schematic design of tire inducible Original iHelper plasmid is shown in Figure 13. The Original iHelper comprises, in the 5' to 3' direction, a first tetracycline inducible promoter operably linked a polynucleotide encoding the adenovirus E4orf6. a second tetracycline inducible promoter operably linked to an adenovirus E2A coding region and an adenovirus VA I/II gene.

[00334] To assess the ability of Original iHelper to support inducible rAAV production, a HEK293 derived cell was transiently transfected with the Original iHelper in combination with one of the iTrans#! through iTrans#8 plasmids and a cis plasmid encoding a GFP-based reporter transgene. The HEK293 derived host cell expressed a tetracycline dependent transactivator capable of activating transcription by the tetracycline inducible promoters used in the Original iHelper and in the iTrans#! through iTrans#8 constructs. Following transfection, the cells were incubated in the presence of 0, 100, 500 or 1,000 ng/ml Doxycycline. rAAV titer produced were measured using digital PCR. The results are shown in Figure 15. In the absence of Doxycycline, rAAV titer produced by the inducible helper and inducible trans plasmid was significantly lower than the titer produced by a constitutive helper in combination with the same inducible trans plasmid. The highest Doxycycline dependent titer was produced by the iTrans#5 and iTrans#7 plasmids.

[00335] To test the contribution of E4 open reading frame polypeptide in this system, 5 additional inducible helper plasmids were prepared. Figure 16. iHelper#!, #2, #3, #4 and #5 differ from the Original iHelper in that they comprise a polynucleotide encoding the adenovirus E4orf6-6/7, E4orf4-6/7, E4orf3-6/7, E4orf2-6/7, and E4orfl -6/7, respectively, operably linked to the first tetracycline inducible promoter.

[00336] The ability of iHelper# 1 through iHclpcr#5 to support inducible rAAV production was assessed as described above. Figure 17. iHelper#3 comprising a polynucleotide encoding E4orf3- 6/7 gave the highest rAAV titer of the construct combinations tested.

Inducible trans plasmids

[00337] Tire inducible iTrans#5 construct was modified to limit Rep polypeptide expression in the uninduced state and to improve rAAV titers. Li et al., J of Virology. 71:5236-5243 (1997). To produce the iTrans#5ATGtoACG construct. iTrans#5 has been modified to change the ATG start codon of the Rep78/68 polypeptide to ACG. Figure 18. In the iTrans#5ptight construct, the Ptight tet promoter was used to control expression of the transcription unit comprising the AAV8 Cap coding region, IRES and AAV2 Rep78/68/52/40 coding region (Loew R, et al. BMC Biotechnol. 2010 Nov 24;10:81. doi: 10.1186/1472-6750-10-81). In the iTrans#5 iTrans#5AU construct an AU rich mRNA destabilizing (AREs) was inserted into the 3' untranslated region of the transcript comprising the AAV8 Cap coding region. IRES and AAV2 Rep78/68/52/40 coding region.

Pharm et aL, Attenuation of leakiness in doxycycline-inducible expression via incorporation of 3' AU-rich mRNA destabilizing elements; BioTechniques 45(2): 155-162 (2008). Tire ability ofthe new⁷ inducible trans constructs to support inducible rAAV production was assessed as described above using tire inducible Original iHelper construct. Figure 18. The experiments using the modified trans constructs comprising the AU rich element or the Ptight tet promoter produced substantially the same rAAV titer as the control experiment using the iTrans#5 construct (Original design in Figure 18). The construct comprising the ATG to ACG mutation at the start codon produced negligible amount of rAAV.

[00338] Exchanging the Rep coding region for a "scrambled" Rep coding region increased rAAV titer. iTrans#5scrambled comprises a scrambled Rep coding region that was created by shuffling synonymous codon pairs while maintaining codon use and the free energy of folded RNA to prevent large changes in secondary structure. Sitaraman et al., PNAS 108(34): 14294-14299 (2011). Tire ability of iTrans#5scramled to support inducible rAAV production was assessed as described above using the inducible Original iHelper construct. Figure 19. Tire rAAV titer produced by iTrans#5 scrambled used in combination with the inducible Original iHelper is more than twice the titer produced by iTrans#5.

[00339] The iTrans#9 construct comprises separate Rep52/40 and Rep78/68 coding regions. As such, iTrans#9 comprises, in a 5’to 3’ direction, (1) a tetracycline inducible promoter operably linked to (2) a Rep cassette comprising, from 5’ to 3’, an AAV2 Rcp52/40 coding region, an IRES and a Rep78/68 coding region, and (3) a CMV promoter operably linked to (4) an AAV8 Cap coding region, wherein the Rep52/40 and Rep78/68 coding regions comprise mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequences encoded, and wherein the Rep78/68 coding region comprises mutation(s) that inactivate the pl9 promoter without altering the Rep polypeptide sequences encoded. The ability of the iTrans#9 and iTrans#5 constructs to support inducible rAAV production was assessed as described above using tire inducible Original iHelper construct (data not shown). While the 2 constructs produced comparable rAAV titers in tire presence of 1,000 ng/ml doxycycline, the iTrans#5 construct produced 2 orders of magnitude lower rAAV titer in the absence of doxycycline than iTrans#9. [00340] The iTrans#7 construct has been re-cloned from a pcDNA3. 1 backbone into a PiggyBac vector and a cHS4 core sequence immediately upstream of the CMV promoter has been removed to create iTrans#7v2. iTrans#v3 was created by replacing the tetracycline inducible promoter of iTrans#7v2 (SEQ ID NO: 2) with a slightly different tetracycline inducible promoter (SEQ ID NO: 2). Figure 20. iTrans#7v3 was further modified to replace the CMV promoter driving Cap expression with a tetracycline inducible promoter to produce the iTrans#7TetCap construct. Inducible trans/helper plasmid set

[00341] The iHelper#6 construct comprises, in the 5' to 3' direction, an adenovirus E2A coding region, an adenovirus L4 22K/33K promoter operably linked to an adenovirus L4 22K/33K coding region, a bidirectional inducible promoter operably linked to the E2A coding region and to an adenovirus E4orf6-6/7 coding region. Figure 21. Inducible helper plasmid was generated to determine whether it can be combined with tire iTrans#! through iTrans#8 plasmids described herein for use in rAAV production.

[00342] iHelper#6-sRep was generated by modifying iHelper#6 to insert a Rep expression cassette at the 5' end of iHelper#6, wherein the cassette comprises, in a 5' to 3' direction, a tetracycline inducible promoter operably linked to an AAV2 Rep52/40 coding region, wherein the AAV2 Rep52/40 coding region comprises mutation(s) that inactivate the p40 promoter without altering the Rep polypeptide sequences encoded. Figure 22. iTrans#10 was generated to complement the iHelper#6-sRep construct. Figure 22. iTrans#10 was derived from iTrans#5 by modifying the AAV2 Rep78/68/52/40 coding region to introduce mutation(s) that inactivate the pl9 promoter without altering the Rep polypeptide sequences encoded, and thereby eliminate Rep52/40 expression from the AAV2 Rep78/68/52/40 coding region. iTrans#10 additionally comprises a hPGK promoter operably linked to a Tet-on 3G coding region, wherein tire tetracycline inducible promoter and the hPGK promoter are positioned adjacent to each other. [00343] The ability of the inducible trans and helper constructs described herein to support inducible rAAV production was assessed as described above. Figure 23. The trans and helper combinations tested are identified in Table 5.

Table 5. Trans and helper construct combinations tested in Figure 23. Tests were performed using a cis plasmid encoding transgene A. Helper#5 (lanes 1-10) does not contain an inducible promoter and has been described in Int’l App. Pub. No. WO 2023/060113, which is incorporated herein by reference in its entirety. pAAV2/8 trans plasmid encodes wild-type AAV2 rep2 and AAV8 cap proteins and does not contain inducible promoters (lanes 1-4 and 11-12). pAAV2/8 trans has been described in Int’l App. Pub. No. WO 2003/052051, which is incorporated herein by reference in its entirety .

EXAMPLE 3. TWO-PLASMID STABLE CELL LINE DEVELOPMENT FOR RECOMBINANT AAV PRODUCTION.

[00344] A two stable transgene based system is developed for producing recombinant AAV particles. The system comprises a first stable packaging transgene encoding trans (i.e., AAV Rep and Cap) functions and helper functions and a second stable cis transgene encoding the rAAV genome. See, e g., Figures 24-26. In some embodiments, the packaging transgene encodes constitutively expressed trans and helper functions. In some embodiments, the packaging transgcnc comprises the packaging plasmids disclosed in Int’l App. Pub. No. WO 2024/211780, which is incorporated herein by reference in its entirety. In some embodiments, the packaging transgene comprises the pHRC #3, #5 or #7 packaging plasmid disclosed in Int’l App. Pub. No. WO 2024/211780. In some embodiments, the packaging transgene encodes inducible trans functions, inducible helper functions, or inducible trans and helper functions. In some embodiments, the inducible trans functions are provided by a polynucleotide described herein, for example, Rep/Cap 5.0 and 5.4 of Figure 2, iTrans#5, #6/7/8, #9, and #10. In some embodiments, the inducible helper function is provided by a polynucleotide described herein, for example. iHelper#6. In some embodiments, the packaging transgene comprises the trans and helper functions depicted in figures 24-26. The packaging transgene and cis transgene are introduced into a host cell suitable for rAAV production. In some embodiments, the host cell endogenously express the E1A and E1B helper genes. In some embodiments, the host cells are HEK293 cells or a cell line derived from HEK293 cells. The packaging transgene and cis transgene can be introduced into a host cell using any method known to a skilled artisan. In some embodiments, the packaging transgene and cis transgene are introduced into the host cell by transfection. In some embodiments, the packaging transgene and cis transgene are introduced into the host cell by site specific recombination. In some embodiments, the packaging transgene and cis transgene are introduced into the by nuclease (e.g., CRISPer/cas9) mediated integration. In some embodiments, the packaging transgene and cis transgene are introduced into the host cell by integrase (e g., BxBbl integrase) mediated integration. In some embodiments, the packaging transgene and cis transgene are introduced into the host cell by integrase (e.g., BxBl integrase) mediated integration, wherein the packaging and cis transgenes are both flanked by integration sites and the host cell (e.g., HEK293 cell) genome comprises integration sites/landing pads at safe harbor loci for insertion of multiple copies of transgenes. In some embodiments, both the packaging transgene and cis transgene are integrated into a safe harbor locus. In some embodiments, the packaging transgene is integrated into a first safe harbor locus. In some embodiments, the cis transgcnc is integrated into a second safe harbor locus.

[00345] While the disclosed methods have been described in connection with what is presently considered to be the most practical and preferred embodiments, it is to be understood that the methods encompassed by the disclosure are not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalent arrangements included within tire spirit and scope of the appended claims.

[00346] All publications, patents, patent applications, internet sites, and accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

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47. Su, W. (2021). Development of a self-silencing adenovirus for the efficient manufacture of adeno-associated virus vectors. PhD Thesis, Tire University of Oxford.

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53. Eisenstein, M. (2020). Enzymatic DNA synthesis enters new phase. Nature Biotechnology’, 38, 1113-1115.

54. Murphy, M., Gomos-Klein, J., Stankic, M., & Falck-Pedersen, E. (2007). Adeno-associated vims type 2 p5 promoter: a rep-regulated DNA switch element functioning in transcription, replication, and site-specific integration. Journal of Virology, 81, 3721-3730.

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Claims

CLAIMS What is claimed is:

1. A recombinant mammalian cell comprising a first and/or second stably integrated polynucleotide wherein a) the first polynucleotide encodes an adenovirus E2A DNA binding protein (DBP). an adenovirus E4 polypeptide and an adenovirus VA RNA gene: and b) the second polynucleotide comprises an AAV rep gene and an AAV cap gene, optionally wherein the first and second polynucleotides are comprised by a packaging polynucleotide.

2. The recombinant cell of claim 1, wherein the first polynucleotide comprises, in a 5' to 3' order, a) a first promoter operably linked to a polynucleotide encoding the adenovirus E4 polypeptide; b) a second promoter operably linked to a polynucleotide encoding the adenovirus E2A DNA binding protein (DBP); and c) a polynucleotide encoding the adenovirus VA RNA gene; wherein the E4 polypeptide comprises the E4 ORF 1-6/7, E4 ORF2-6/7, E4 ORF3-6/7, E4 0RF4- 6/7, E4 ORF6-6/7, E4 0RF6, or E4 ORF3+6-6/7.

3. The recombinant cell of claim 2, wherein the E4 polypeptide comprises the E4 ORF6-6/7, E4 0RF6, or E4 ORF3+6-6/7.

4. The recombinant cell of claim 2, wherein the E4 polypeptide comprises the E4 ORF6 or E4 ORF6/7.

5. The recombinant cell of claim 2 to claim 4, wherein the E4 0RF1, 0RF2, 0RF3, ORF4, 0RF6 and ORF 6/7 polypeptide comprises an amino acid sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10-14 and 15, respectively.

6. The recombinant cell of claim 2 to claim 4, wherein the polynucleotide encoding the adenovirus E4 polypeptide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16-21 or 22.

7. Tire recombinant cell of claim 6, wherein the polynucleotide encodes the adenovirus E4 OR6 or ORF6-6/7 polypeptide and comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16 or 21.

8. The recombinant cell of any one of claim 2 to claim 7, wherein the first promoter is an inducible promoter.

9. The recombinant cell of claim 8, wherein the inducible promoter is a tetracycline inducible promoter.

10. Tire recombinant cell of claim 9, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

11. The recombinant cell of any one of claim 2 to claim 10, wherein the E2A DBP comprises an amino acid sequence having at least about 90%. 95%. 97%. 98%, 99% or 100% identity to SEQ ID NO: 8.

12. The recombinant cell of any one of claim 2 to claim 10, wherein the polynucleotide encoding the adenovirus E2A DBP comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6 or 7.

13. The recombinant cell of any one of claim 2 to claim 12, wherein the second promoter is an inducible promoter.

14. The recombinant cell of claim 13, wherein the inducible promoter is a tetracycline inducible promoter.

15. Tire recombinant cell of claim 14, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%. 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

16. The recombinant cell of any one of claim 2 to claim 15. wherein the first and second promoters are different.

17. The recombinant cell of any one of claim 2 to claim 15, wherein the first and second promoters are the same, optionally wherein the first and second promoters comprise a bidirectional promoter.

18. The recombinant cell of any one of claim 2 to claim 17, wherein the first polynucleotide further comprises a promoter operably linked to an adenovirus L4 22K/33K polypeptide coding region.

19. The recombinant cell of claim 18, wherein the promoter operably linked to the adenovirus L4 22K/33K polypeptide coding region is an adenovirus L4 22K/33K promoter or an inducible promoter, optionally a tetracycline inducible promoter.

20. The recombinant cell of any one of claim 2 to claim 19, wherein the polynucleotide encoding the adenovirus VA RNA gene comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 30.

21. The recombinant cell of any one of claim 2 to claim 20, wherein the first polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 33-38 or 39.

22. The recombinant cell of any one of claim 1 to claim 21, wherein the second polynucleotide comprises a third promoter operably linked to a third polynucleotide comprising, in 5' to 3' order an AAV cap coding region, an internal ribosome entry site (IRES), and a first AAV rep coding region, wherein a) the AAV cap coding region encodes a VP1. VP2 and VP3 polypeptide; b) the first AAV rep coding region encodes one or more of a Rep78. Rep68. Rep52 and Rep40 polypeptide; c) the first AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity.

23. Tire recombinant cell of claim 22, wherein the first AAV rep coding region encodes a (i) a Rep 78 and Rep 52 polypeptide, (ii) Rep 68 and Rep 40 polypeptide, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptide, wherein the first AAV rep coding region comprises a functional p!9 promoter.

24. The recombinant cell of claim 22, wherein the first AAV rep coding region encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide and wherein the AAV rep coding region comprises a functional p!9 promoter.

25. The recombinant cell of claim 22, wherein tire first AAV rep coding region encodes a (i) a Rep78 polypeptide, (ii) Rep 68, or (iii) a Rep78 and Rep 68 polypeptide and wherein the first AAV rep coding region comprises a pl9 promoter comprising a mutation that reduces or eliminates its activity.

26. Tire recombinant cell of claim 22, wherein the first AAV rep coding region encodes a Rep78 and Rep 68 polypeptide.

27. The recombinant cell of any one of claim 22 to claim 26, further comprising a seventh polynucleotide comprising a seventh promoter and a second Rep coding region, wherein the second Rep coding region encodes (i) a Rep 52 polypeptide, (ii) Rep40 polypeptide or (iii) a Rep52 and Rep40 polypeptide, wherein the second rep coding region comprises ap40 promoter comprising a mutation that reduces or eliminates its activity.

28. The recombinant cell of claim 27, wherein the second Rep coding region encodes a Rep52 and Rep40 polypeptide.

29. The recombinant cell of claim 27 or claim 28. wherein seventh promoter is an inducible promoter.

30. The recombinant cell of claim 29, wherein the inducible promoter is a tetracycline inducible promoter.

31. The recombinant cell of claim 35, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

32. Tire recombinant cell of any one of claim 22 to claim 31, wherein the third promoter is an inducible promoter.

33. The recombinant cell of claim 32. wherein tire inducible promoter is a tetracycline inducible promoter.

34. The recombinant cell of claim 33, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2 or 3.

35. The recombinant cell of any one of claim 22 to claim 34, wherein the IRES comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 69.

36. The recombinant cell of any one of claim 22 to claim 45, wherein the third polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 70 or 72.

37. The recombinant cell of any one of claim 22 to claim 36, wherein the second polynucleotide comprises a nucleotide sequence having at least about 90%. 95%. 97%. 98%, 99% or 100% identity to SEQ ID NO: 71 or 73.

38. The recombinant cell of any one of claim 1 to claim 21, wherein the second polynucleotide comprises a) a fourth promoter operably linked to a fourth polynucleotide comprising in 5' to 3' order i. a polynucleotide encoding the 5' portion of an AAV Rep78 and/or 68 polypeptide, ii. an intron comprising a fifth promoter, and iii. a fifth polynucleotide comprising overlapping coding regions encoding the 3' portion of tire AAV Rep78 and/or 68 polypeptide and an AAV Rep52 and/or Rep40 polypeptide, wherein

- tire fifth promoter is operably linked to the fifth polynucleotide;

- the fourth polynucleotide encodes (i) a Rep78 and Rep52 polypeptides; (ii) a Rep68 and Rep40 polypeptides, or (iii) a Rep78, Rep68, Rep52 and Rep40 polypeptides, - and each of the p!9 and p40 promoters comprises a mutation that reduces or eliminates their activity; and b) a sixth promoter operably linked to a polynucleotide comprising a AAV cap coding region, wherein the AAV cap coding region encodes a VP1, VP2 and VP3 polypeptide.

39. The recombinant cell of claim 38, wherein tire fourth polynucleotide encodes a Rep78 and Rep52 polypeptides.

40. The recombinant cell of claim 38 or claim 39, wherein the fourth promoter is an inducible promoter.

41. Tire recombinant cell of claim 40, wherein the inducible promoter is a tetracycline inducible promoter.

42. The recombinant cell of claim 46. wherein tire inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 2.

43. Tire recombinant cell of any one of claim 38 to claim 42, wherein the fifth promoter is an inducible promoter.

44. The recombinant cell of claim 43, wherein the inducible promoter is a tetracycline inducible promoter.

45. The recombinant cell of claim 49, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 2.

46. Tire recombinant cell of any one of claim 38 to claim 45, wherein tire fifth and sixth promoters are different.

47. The recombinant cell of any one of claim 38 to claim 45, wherein the fifth and sixth promoters are the same.

48. The recombinant cell of any one of claim 38 to claim 47, wherein the intron comprising the fifth promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 57, 58, 59 or 60.

49. The recombinant cell of any one of claim 38 to claim 48, wherein the fourth polynucleotide comprises a nucleotide sequence having at least about 90%. 95%. 97%, 98%, 99% or 100% identity to SEQ ID NO: 61, 62, 63 or 64.

50. The recombinant cell of any one of claim 38 to claim 49, wherein the sixth promoter is an inducible promoter.

51. Tire recombinant cell of claim 50, wherein the inducible promoter is a tetracycline inducible promoter.

52. The recombinant cell of claim 50, wherein the inducible promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 2.

53. Tire recombinant cell of any one of claim 38 to claim 49, wherein tire sixth promoter is constitutive promoter.

54. The recombinant cell of claim 53. wherein tire sixth promoter is a CMV promoter.

55. The recombinant cell of claim 54, wherein the sixth promoter comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4 or 76.

56. Tire recombinant cell of any one of claim 38 to claim 55, wherein the second polynucleotide comprises a nucleotide sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 77, 78, 79, 80, 81 or 82.

57. The recombinant cell of any one of claim 1 to claim 21. wherein tire second polynucleotide comprises in 5' to 3' order an eighth promoter operably linked to a first AAV Rep coding region and a ninth promoter operably linked to an AAV Cap coding region, wherein a) the first AAV rep coding region comprises a functional p 19 promoter and encodes a Rep78, Rep68, Rep52 and Rep40 polypeptide; b) the first AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity; and c) the AAV cap coding region comprises an AAV Cap intron and encodes a VP1, VP2 and VP3 polypeptide.

58. Tire recombinant cell of claim 57, wherein the second polynucleotide comprises in 5' to 3' order an eighth promoter operably linked to a first AAV Rep coding region, a ninth promoter operably linked to an AAV Cap coding region and a tenth promoter operably linked to a second AAV Rep coding region, wherein the second AAV Rep coding region encodes a Rep52 and Rep40 polypeptide, and wherein second AAV rep coding region comprises a p40 promoter comprising a mutation that reduces or eliminates its activity.

59. Tire recombinant cell of claim 57 or claim 58, wherein the one or more of the first AAV Rep coding region, second AAV Rep coding region and AAV Cap coding region comprises a poly A signal.

60. The recombinant cell of any one of claim 57 to claim 58, wherein one or more of the eighth, ninth and tenth promoter is an inducible promoter.

61. The recombinant cell of claim 60, wherein the inducible promoter is a tetracycline inducible promoter.

62. The recombinant cell of any one of claim 1 to claim 61, wherein the AAV rep gene and the AAV cap gene have the same serotype.

63. Tire recombinant cell of any one of claim 1 to claim 61, wherein the AAV rep gene and the AAV cap gene have different serotypes.

64. The recombinant cell of any one of claim 1 to claim 63, wherein the AAV rep gene comprises an AAV2 rep gene.

65. The recombinant cell of any one of claim 1 to claim 64, wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, 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.

66. Tire recombinant cell of claim 65, wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37.

67. The recombinant cell of claim 65. wherein the AAV cap gene comprises a serotype selected from the group consisting of AAV8 or AAV9 serotype.

68. The recombinant cell of claim 65, wherein the AAV cap gene comprises the AAV2 serotype.

69. Tire recombinant cell of claim 65, wherein the AAV cap gene comprises the AAV6 serotype.

70. The recombinant cell of claim 65, wherein the AAV cap gene comprises tire AAV8 serotype.

71. The recombinant cell of claim 65, wherein tire AAV cap gene comprises tire AAV9 serotype.

72. The recombinant cell of any one of claim 1 to claim 71 comprising the first polynucleotide.

73. The recombinant cell of any one of claim 1 to claim 71 comprising the second polynucleotide.

74. The recombinant cell of any one of claim 1 to claim 71 comprising the first and second polynucleotides.

75. The recombinant cell of claim 74, further comprising a sixth polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell.

76. The recombinant cell of claim 75, wherein the sixth polynucleotide is stably integrated.

77. The recombinant cell of any one of claim 1 to claim 76, wherein the cell is a HEK293 cell, HEK293 derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, or PerC6 cell.

78. Tire recombinant cell of claim 77, wherein the cell is a EIEK293 cell or HEK293 derived cell.

79. The recombinant cell of any one of claims 1 to 78, wherein the first and second polynucleotides are comprised by a packaging polynucleotide.

80. The recombinant cell of claim 79, wherein the first polynucleotide comprises any one of SEQ ID NO: 48 and 93-99 and tire second polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-91.

81. Tire recombinant cell of claim 79, wherein the first polynucleotide comprises SEQ ID NO: 48 and the second polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-91.

82. The recombinant cell of claim 79. wherein tire first polynucleotide comprises SEQ ID NO: 48 and the second polynucleotide comprises any one of SEQ ID NO: 70-73 and 77-82.

83. The recombinant cell of any one of claims 79 to 82, wherein the packaging polynucleotide further comprises flanking recognition sites for an integrase or recombinase such that tire packaging polynucleotide can be integrated into a genomic landing site of the host cell comprising the complementary recognition site.

84. The recombinant cell of any one of claims 79 to 83. comprising a sixth polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell.

85. The recombinant cell of claim 84, wherein the sixth polynucleotide is stably integrated.

86. A recombinant cell comprising a stably integrated packaging polynucleotide comprising in a 5 'to 3’ direction.

(1) an inducible promoter operably linked to

(3) a CMV promoter operably linked to

(4) an AAV Cap coding region, operably linked to

(5) a Helper cassette comprising, from 5’ to 3’.

(a) a polynucleotide encoding an adenovirus E2A DNA binding protein (DBP),

(b) a polynucleotide comprising a gene encoding an adenovirus L4 22K/33K polypeptide, (c) an inducible promoter operably linked to a polynucleotide encoding an adenovirus E4 orf6-6/7 polypeptide, optionally wherein the inducible promoter is a tetracycline inducible promoter, and

(d) a polynucleotide encoding an adenovirus VA RNA gene, wherein the 5' to 3' orientation of both tire polynucleotide encoding the E2A DBP and the polynucleotide encoding the VA RNA are opposite to tire 5' to 3' orientation of the adenovirus L4 22K/33K polynucleotide and the E4 orf polynucleotide.

87. The recombinant cell of claim 86, wherein tire gene encoding the adenovirus L4 22K/33K polypeptide comprises an L4 22K/33K promoter operably linked to an L4 22K/33K coding region.

88. The recombinant cell of claim 86, wherein the gene encoding the adenovirus L4 22K/33K polypeptide comprises an inducible promoter operably linked to an L4 22K733K coding region, optionally wherein the inducible promoter is a tetracycline inducible promoter.

89. The recombinant cell of any one of claims 86 to 88, wherein the packaging polynucleotide further comprises flanking recognition sites for an integrase or recombinase such that the packaging polynucleotide can be integrated into a genomic landing site of the host cell comprising the complementary recognition site.

90. The recombinant cel of any one of claims 86 to 88, wherein the cell further comprises a stably integrated polynucleotide comprising in a 5 'to 3’ direction, a) a 5' AAV2 inverted terminal repeat sequence (5’ ITR). b) a promoter operably linked to a transgene coding region encoding a gene product, optionally wherein the promoter is a tissue-specific promoter to drive transgene expression in a target tissue cell, and c) a 3’ AAV2 inverted terminal repeat sequence (5’ HR).

91. A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of claim 72; b) introducing into tire cell i. a polynucleotide encoding an AAV capsid protein; ii. a polynucleotide encoding a functional rep gene; and iii. a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (1TR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell, and c) maintaining the cell under conditions that allow production of the rAAV particles.

92. A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of claim 73; b) introducing into the cell i. a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (1TR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; ii. a polynucleotide comprising sufficient helper functions to permit packaging of the genome into the AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid, and c) maintaining the cell under conditions that allow production of tire rAAV particles.

93. A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of claim 74; b) introducing into the cell a polynucleotide comprising a genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a target cell; and c) maintaining the cell under conditions that allow production of the rAAV particles.

94. A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of claim 75 or claim 76; and b) maintaining the cell under conditions that allow production of the rAAV particles.

95. A method of producing rAAV particles, comprising a) providing a cell culture comprising the recombinant cell of any one of claims 84, 86 and 90; and b) maintaining the cell under conditions that allow production of tire rAAV particles.

96. Tire method of any one of claims 91 to 95, wherein the cell culture is a suspension culture.

97. Tire method of any one of claims 91 to 96, further comprising recovering the rAAV particles.

98. The method of any one of claims 91 to 97, wherein the cell culture has a volume between about 50 liters and about 20,000 liters.

99. Tire method of any one of claims 91 to 98, wherein the gene product is a polypeptide or a double stranded RNA molecule.

100. The method of claim 99, wherein the gene product is a polypeptide.

101. The method of claim 100. wherein tire 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 nonmembrane associated splice variant of VEGF receptor 1 (sFlt-1).

102. Tire method of claim 100, wherein the gene product is an gamma-sarcoglycan, Rab Escort Protein 1 (REP1/CHM). retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated 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), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6- phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alphaglucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1 -antitr psin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1). survival motor neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3). porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgGl) Fc fusion.

103. The method of claim 100, wherein tire gene product is a dystrophin or a microdystrophin.

104. The method of claim 99, wherein the gene product is a microRNA, snRNA or an antisense RNA.