Compositions and methods for increasing aav productivity

Abstract

Provided are vectors and vector systems for the production of recombinant adeno-associated viral vectors, as well as methods of using the vectors and vector systems to optimize productivity.

Description

COMPOSITIONS AND METHODS FOR INCREASING AAV PRODUCTIVITYBACKGROUND

[0001] Adeno-associated viral (AAV) vectors are commonly used for nucleic acid delivery into cells. Successes in AAV-mediated gene replacement, gene silencing, and gene editing make AAV a desirable therapeutic vector, with AAV-based therapeutics gaining regulatory approval in Europe and the United States. However, existing manufacturing systems can be difficult to optimize and often produce insufficient quantities of vector genomes for gene therapy applications. Accordingly, improved AAV vector manufacturing systems are needed.SUMMARY OF THE INVENTION

[0002] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Example, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various ways, and all such combinations are intended to fall within the scope of the present invention.

[0003] This disclosure relates to vectors comprising one or more modified adenoviral nucleic acids for the production of recombinant adeno-associated virus (rAAV). In some embodiments, the vector comprises nucleic acid E4 comprising a deletion, nucleic acid E2a, and a virus-associated (VA) RNA from an adenovirus genome. In some embodiments, the nucleic acid E2a is a modified nucleic acid E2, e.g., a nucleic acid E2 comprising a deletion.

[0004] In some embodiments, the vector comprises nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein nucleic acid E4 comprises a deletion of open reading frames 1-3 (orf 1-3).

[0005] In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs, for example, a deletion of at least 1000 base pairs in E4orfl-E4orf3, or a deletion of at least 1400 base pairs in E4orfl-E4orf4. In some embodiments, the nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4; or a nucleic acid sequence having at last 85% identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4.

[0006] In some embodiments, the nucleic acid E4 comprises open reading frame 6 (orf6), i.e., nucleic acid E4 comprising a deletion of orfs 1-4 and 7. In some embodiments, the nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid sequence having at last 85% identity to SEQ ID NO: 3.

[0007] In some embodiments, the nucleic acid E4 comprises E4orf6 and E4orf7, i.e., nucleic acid E4 comprising a deletion of orfs 1-5. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NON or a nucleic acid sequence having at last 85% identity to SEQ ID NON.

[0008] In some embodiments, the vector further comprises an E2a deletion. In some embodiments, the E2 deletion comprises a deletion of greater than about 400 base pairs in the 5’ untranslated region of the E2a gene. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 or a nucleic acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0009] In some embodiments, the vector comprises a nucleic acid sequence of SEQ ID NO: 13, 14, 15, 16, 17 or 18 or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 13, 14, 15, 16, 17 or 18.

[0010] Also provided is a vector system comprising at least two vectors, wherein a first vector comprises a helper plasmid, wherein the helper plasmid is a plasmid comprising a modified E4 nucleic acid, an E2a nucleic acid, and a virus-associated (VA) RNA from an adenovirus genome, described herein. In some embodiments, the second vector is a plasmid comprising a transgene.

[0011] Also provided is a vector system comprising a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein the at least a portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids, as compared to production using a vector system lacking the nucleic acid E4 deletion. In some embodiments, the at least a portion of nucleic acid E2a comprises a deletion.

[0012] Further provided is a vector system comprising at least two vectors, wherein a first vector comprises one or more of a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein the at least a portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids, as compared to production using a vector systemlacking the nucleic acid E4 deletion. In some embodiments, the at least a portion of nucleic acid E2a comprises a deletion. Optionally the first vector comprises the portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome.

[0013] In some embodiments, the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both as compared to a control AAV product produced by an AAV vector system lacking deletion of nucleic acids E4 or lacking deletions of nucleic acid E4 and nucleic acid E2a. In some embodiments, the second vector comprises a transgene.

[0014] Also provided is a method of producing an AAV product comprising an AAV capsid. The method comprises introducing into a host cell any of the vector systems described herein, incubating the host cells under culture conditions that promote AAV product production, and purifying the AAV product.

[0015] Another aspect of the invention is a method of delivering a transgene to a cell, the method comprising introducing into the cell an AAV product produced by the provided methods. In some embodiments, the method is an in vitro method. In some embodiments, the transgene is expressed by the cell. Some embodiments provide the use of an AAV product of the disclosure in a method of delivering a transgene to a cell, wherein the transgene is optionally expressed by the cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0017] FIG. l is a schematic illustrating a vector construct comprising helper genes E2a, E4, and VA RNA. The E2a gene is about 5.3 kilobase (kb) and is regulated by the E2E promoter to generate a gene product. E2a and L4 gene overlap on opposite strands, which resulted in L4 regions being included in the helper plasmids. The E4 gene contains six open reading frames (orf) and is about 3.2 Kb. The VA (viral associated) RNA gene is about 0.74 Kb.

[0018] FIG. 2 shows exemplary helper vector constructs comprising from the top (a) VA RNA, a nucleic acid E4, a nucleic acid E2a and an adenoviral backbone without deletions (designated control); (b) VA RNA, a nucleic acid E4 comprising E4 deletion 1 (i.e., nucleic acid E4 comprising a deletion of E4orfl-E4orf3), a nucleic acid E2a, and an adenoviral backbone comprising a deletion (designated del-1); (c) VA RNA, an E4 nucleic acid comprising E4 deletion 2 (i.e., nucleic acid E4 comprising deletion of E4orf 1 -E4orf4, having only E4orf6-7), and an adenoviral backbone comprising a deletion (designated del-2); (d) VA RNA, an E4 nucleic acid, an E2a nucleic acid comprising a deletion, and an adenoviral backbone comprising a deletion (designated del-3); (e) VA RNA, a nucleic acid E4 comprising E4 deletion 1 (described above), an E2a nucleic acid comprising a deletion (a deletion of about 900 base pairs), and an adenoviral backbone comprising a deletion (designated del-4); and (f) VA RNA, an E4 nucleic acid comprising E4 deletion 2 (described above), an E2a nucleic acid comprising a deletion and an adenoviral backbone comprising a deletion (designated del-5). The backbone deletion comprised deletion of nucleic acid residues or regions thereof that were not required for functional plasmid formation.

[0019] FIG. 3 provides graphs showing the effects of the various helper plasmids described in FIG. 2 on viral genome (VG) concentration (left) and capsid concentration (right) using AAV9 serotype and a GFP / Luciferase genome. The plasmids with E4 deletions all increased production as evidenced by higher concentrations of VG and capsid per liter as compared to production by control plasmids or plasmids lacking an E4 deletion (e.g., having an E2a deletion and a backbone deletion, del-3). VG titer was measured from crude lysates by ddPCR and capsid titer was measured by capsid ELISA.

[0020] FIG. 4 is a graph showing the effects of the control plasmid and the helper plasmid comprising an E2a nucleic acid deletion, an E4 nucleic acid with deletion 2, a VA RNA and an adenoviral backbone deletion (del-5), in multiple AAV serotypes, on VG concentration (left) and AAV capsid concentration (right). For each serotype, an increase in viral genome and capsid production was observed using the plasmid designated del-5 as compared to the control plasmid. VG titer was measured from crude lysates by ddPCR and capsid titer was measured by capsid ELISA.DETAILED DESCRIPTION

[0021] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope ofthe compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

[0022] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0023] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.Definitions

[0024] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0025] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0026] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include 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 include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0027] Wherever embodiments are described with the language “comprising” or “having,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0028] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number.Vectors

[0029] Provided herein are vectors comprising one or more modified adenoviral nucleic acids. In some embodiments, the vector comprises a nucleic acid E4, a nucleic acid E2a, and a virus-associated (VA) RNA from an adenovirus genome, wherein the nucleic acid E4 comprises a deletion.

[0030] As used throughout, a vector refers to a nucleic acid construct. Any nucleic acid construct that comprises one or more elements required for AAV production can be used as any of the vectors or as a component of the vector systems described herein. Suitable vectors, include, without limitation, plasmids, minimal vectors (e.g., minicircles, Nanoplasmids™, doggybones, MIDGE vectors, and the like), viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. Suitable DNA minimal vectors include, without limitation, linear covalently closed DNA (e.g., ministring DNA), linear covalently closed dumbbell shaped DNA (e.g., doggybone DNA, dumbbell DNA), minicircles, Nanoplasmids™, minimalistic immunologically defined gene expression (MIDGE) vectors, and others known to those of skill in the art. DNA minimal vectors and their methods of production are described in, e.g., U.S. Patent Application Publication Nos. 20100233814, 20120282283, 20130216562, 20150218565, 20150218586, 20160008488, 20160215296, 20160355827, 20190185924, 20200277624, and 20210010021, all of which are herein incorporated by reference in their entireties. In some embodiments, the vector is a circular, single stranded or double stranded nucleic acid sequence construct, e.g., a double-stranded DNA plasmid. In some cases, a vector is an extrachromosomal circular DNA comprising one or more origins of replication capable of autonomous replication in a given cell, for example, a eukaryotic cell or a bacterial cell. In some embodiments, the vector does not comprise one or more sequences necessary forautonomous replication in bacterial cells, for example, a bacterial origin of replication. In some embodiments, a vector comprises the one or more elements required for AAV replication. In some embodiments, a vector system comprises a vector or combination of vectors comprising the elements required for AAV replication. In some embodiments, a vector system comprises a vector comprising the elements required for AAV replication and a vector comprising a gene or interest.

[0031] As used herein, the term nucleic acid or nucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference nucleic acid. A nucleic acid sequence can comprise combinations of deoxyribonucleic acids and ribonucleic acids. Such deoxyribonucleic acids and ribonucleic acids include both naturally occurring molecules and synthetic analogues. Nucleic acids also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0032] In some embodiments, the nucleic acids in the vectors described herein are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and / or elimination of mRNA instability elements. Methods to generate optimized polynucleotides for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly, all of which are herein incorporated by reference in their entireties. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In certain embodiments, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and / or function as the original amino acid.

[0033] As used throughout, a nucleic acid E4 can comprise a coding and / or a non-coding sequence from the E4 gene of an adenoviral genome. A gene can include exonic regions, intronic regions, and / or untranslated regions from a genomic sequence. In some cases, agene, for example, E4, may have one or more coding sequences due to alternative splicing or alternative translation initiation, etc. A coding sequence may be a wild-type or a non-naturally occurring coding sequence (e.g., a codon optimized E4 coding sequence).

[0034] In some cases, a nucleic acid E4 encodes one or more E4 gene products, for example, one or more E4 proteins or fragments thereof. The one or more E4 gene products or fragments thereof can be encoded by one or more open reading frames (orf) (i.e., orfs 1-7) of an adenoviral E4 genomic sequence. One or more E4 gene products or fragments thereof can modulate transcription, the cell-cycle, cell signaling and / or DNA repair. For example, E4orfl encodes a protein that interacts with cellular factors comprising a PDZ domain binding motif. E4orf3 encodes an 1 IkDa protein that is functionally redundant with the 34kD protein encoded by E4orf6. The E4orf3 protein can be involved in viral DNA replication, late viral protein synthesis, shut-off of host protein synthesis, and / or virus production. E4orf4 encodes a protein that can regulate protein phosphorylation during infection through its ability to bind to protein phosphatase 2A (PP2A), one of the major serine / threonine-specific phosphatases in the cell. E4orf6 encodes a 34 kDa protein that forms a multi-functional complex involved in viral DNA replication, RNA processing, nucleo-cytoplasmic transport of late viral mRNA, and the shut-off of host protein synthesis. The gene product of E4orf6 and E4orf7 is a fusion of E4orf6 and E4orf7 proteins. For example, in AdV type 5, the protein consists of 58 residues from the amino-terminus of E4orf6, fused to the 92 amino acids of E4orf7. The E4orf6 / 7 protein modulates transcriptional activity through its interaction with the cellular E2F / DP family of cellular transcription factors.

[0035] As used throughout, a nucleic acid E2a can comprise a coding and / or a non-coding sequence from the E2a gene of an adenoviral genome. A nucleic acid E2a can comprise exonic regions, intronic regions, and / or untranslated regions from a genomic E2a sequence. In some cases, a nucleic acid E2a encodes an E2a protein or a fragment thereof. E2A is a single-stranded DNA binding protein that stimulates viral DNA replication and gene transcription.

[0036] As used herein, VA RNA is an RNA that functions in inhibiting the cellular innate immune protein double-stranded RNA-activated kinase (PKR), the inhibition of which ensures efficient virus protein synthesis. VA RNA has also been shown to facilitate the synthesis and assembly of AAV structural proteins. It will be readily appreciated to thoseof skill in the art that the VA RNA nucleic acid sequence is a non-translated nucleic acid sequence that gives rise to the VA RNA.

[0037] The vectors described herein can function as helper plasmids in a plasmid system for producing recombinant AAV capsids. Typically, adenoviral (AdV) helper factors El A, E1B, E2A, E4, and VA RNA are necessary for viral replication. Any of the helper virus factors described herein can be from an adenoviral (AdV) genome, for example, from an AdV type 2 genome or from an AdV type 5 genome. It is understood that, if a nucleic acid sequence for an AdV type 2 helper virus factor, for example, E4, E2a or VA, is provided herein, the corresponding AdV type 5 nucleic acid sequence is also provided. Sequences provided herein correspond to AdV type 2, but comparable sequences can be derived for other types of AdV. Nucleic acids encoding these AdV helper factors or a portion thereof (i.e., El A, E1B, E2A, E4, and VA RNA) can be transfected into a host cell on a single plasmid. Alternatively, one or more plasmids comprising these helper factors can be transfected into a host cell. The helper factors can also be expressed by transfecting, into a host cell, one or more plasmids encoding helper factors that are not endogenously expressed by the host cell. In some instances, certain host cells such as, for example, HEK293T cells, endogenously provide some, but not all, required helper factors, and the remaining helper factors can be provided exogenously via plasmid transfection. For example, HEK293T cells endogenously express AdV El A and E1B genes and can be transfected with any of the vectors, e.g., helper plasmids, described herein to provide at least the minimum helper elements required for AAV replication, for example, one or more AdV E4 gene products or fragments thereof, E2a gene products or fragments thereof, and virus-associated (VA) RNA to the host cell, to produce the required helper elements for AAV production, e.g., El A, E1B, E2A, one or more E4 gene products, and VA RNA in the host cell.

[0038] In any of the helper plasmids described herein, a nucleic acid sequence encoding one or more of the helper factors or a functional portion thereof for AAv replication, e.g., an E4 gene product, an E2a gene product, and / or VA RNA, can be operably linked to a transcriptional regulatory element that controls the expression of the helper factor. In certain embodiments, the transcriptional regulatory element comprises a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a native promoter. Suitable promoters are known to those of skill in the art and include, without limitation, an RSV LTR promoter, a CMV immediate early promoter, an SV40promoter, a dihydrofolate reductase promoter, a cytoplasmic P-actin promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionine (MT) promoter, a mouse mammary tumor virus (MMTV) promoter, a T7 promoter, an ecdysone insect promoter, a tetracycline-repressible promoter, a tetracycline-inducible promoter, an RU486-inducible promoter, and a rapamycin-inducible promoter.

[0039] In some embodiments, the vector comprises nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein nucleic acid E4 comprises a deletion of one or more E4 open reading frames (orf). As used throughout, deletion of an orf refers to deletion or removal of at least a portion of an E4orf such that reduced expression and / or activity of one or more gene products encoded by the E4orf results. As used herein, a reduction can be a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the expression and / or activity. In some examples, the E4 deletion results in reduced expression and / or activity of one or more E4 gene products selected from the group consisting of E4orfl, E4orf2, E4orf3, E4orf4, and E4orf6 / 7.

[0040] In some embodiments, the nucleic acid E4 deletion is a deletion of open reading frames 1-3 (E4orfl-3), i.e., the nucleic acid E4 deletion comprises E4orf4, Eorf6, and Eorf7. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, or 1075 base pairs) of a nucleic acid sequence comprising E4orfl-E4orf3 (SEQ ID NO: 2). In some embodiments, the nucleic acid E4 comprising a deletion of E4orfsl-3 comprises SEQ ID NO: 1 or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 1.

[0041] In some embodiments, the vector comprises orf6 of nucleic acid E4; nucleic acid E2a or a portion thereof; and VA RNA of an adenoviral genome, wherein nucleic acid E4 comprises a deletion.

[0042] In some embodiments, the nucleic acid E4 comprising E4orf6 and / or E4orf7 comprises a deletion of one or more orfs selected from the group consisting of E4orfl, E4orf2, and E4orf4, for example, a deletion of E4orfsl-4. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350 or 1400 base pairs) of a nucleic acid sequence comprising deletion of E4orf 1 -E4orf4 (SEQ ID NO: 5). An exemplary nucleotide sequence comprising a nucleic acid comprisingE4orf6, a nucleic acid comprising E4orf7, and a nucleic acid E4 deletion of E4orfsl-5 is set forth herein as SEQ ID NO: 4. In some embodiments, the nucleic acid E4 comprising a nucleic acid comprising E4orf6, a nucleic acid comprising E4orf7, and a nucleic acid E4 deletion of E4orfsl-5 has at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 4.

[0043] In some embodiments, the vector comprises a nucleic acid E4 comprising a deletion, as described above, and a nucleic acid E2a that does not comprise a deletion, i.e., a wildtype adenoviral nucleic acid E2a. Exemplary wildtype nucleic acid E4 and wildtype nucleic acid E2a sequences are set forth herein as SEQ ID NOs: 6 and 7, respectively.

[0044] In some embodiments, the vector comprises a nucleic acid E4 comprising a deletion, as described above, and a nucleic acid E2a comprising a deletion. In some embodiments, the E2a deletion comprises a deletion of at least about 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 base pairs in an untranslated genomic region of the nucleic acid E2a, for example, a 5’ untranslated region (UTR). Exemplary nucleic acid sequences comprising a nucleic acid E2a comprising a deletion of at least 500 base pairs in the 5’ UTR are set forth herein as SEQ ID NOs: 8-12. SEQ ID NO: 8 is a nucleic acid E2a comprising about a 2 kb deletion in the 5’ UTR. SEQ ID NO: 9 is a nucleic acid E2a comprising about a 1 kb deletion in the 5’ UTR. SEQ ID NO: 10 is a nucleic acid E2a comprising about a 1.4 kb deletion in the 5’ UTR. SEQ ID NO: 11 is a nucleic acid E2a comprising about a 500 base pair deletion in the 5’ UTR. SEQ ID NO: 12 is a nucleic acid E2a comprising about a 900 base pair deletion in the 5’ UTR.

[0045] Any of the vectors described herein can further comprise a nucleotide sequence encoding VA RNA, for example, a nucleotide sequence comprising SEQ ID NO: 13 or a nucleotide sequence comprising a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 13.

[0046] In some embodiments, the vector comprises nucleic acid E4 comprising a deletion (e.g., SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4), a nucleic acid E2a (e.g., SEQ ID NO: 7 or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:7), and VA RNA (e.g., SEQ ID NO: 13 or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 13).

[0047] In some embodiments, the vector comprises nucleic acid E4 comprising a deletion (e.g., SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4), a nucleic acid E2a with a deletion (e.g., SEQ ID NO: 8, 9, 10, 11, or 12; or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 8, 9, 10, 11, or 12) and VA RNA (e.g., SEQ ID NO: 13 or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 13).

[0048] An exemplary vector sequence (plasmid pOX-02010) comprising SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13 is set forth herein as SEQ ID NO: 14. An exemplary vector sequence (plasmid pOX-02011) comprising SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 13 is set forth herein is SEQ ID NO: 15.

[0049] In some embodiments, the vector comprises nucleic acid E4 comprising a deletion (e.g., SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4), nucleic acid E2a comprising a deletion (SEQ ID NO: 8, 9, 10, 11, or 12), and VA RNA (SEQ ID NO: 13). An exemplary vector sequence (plasmid pOX-02013) comprising SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 13 is set forth herein as SEQ ID NO: 16. An exemplary vector sequence (plasmid pOX-02014) comprising SEQ ID NO: 4, SEQ ID NO: 12 and SEQ ID NO: 13 is set forth herein is SEQ ID NO: 17.

[0050] In some embodiments, the vector comprises nucleic acid E4 without a deletion (e.g., SEQ ID NO: 6), nucleic acid E2a comprising a deletion (SEQ ID NO: 8, 9, 10, 11 or 12), and VA RNA (SEQ ID NO: 13). An exemplary vector sequence (plasmid pOX-02012) comprising SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 13 is set forth herein is SEQ ID NO: 18.

[0051] Any of the vectors described herein, for example, a helper plasmid, can comprise a backbone nucleic acid sequence comprising one or more regulatory elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), an origin(s) of replication, and / or a selectable marker gene). In some embodiments, the helper plasmid vector comprises a backbone nucleic acid sequence without a deletion or a backbone nucleic acid sequence comprising a deletion.

[0052] Any of the vectors provided herein can comprise a nucleic acid sequence having at least 60%, 65%, 70%, 80%, 90%, 91%, 93%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequences described herein (e.g., SEQ ID NOs: 1-18). The term identity as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence, for example, any one of SEQ ID NOs: 1-18. Alternatively, percent identity can be any integer from 60% to 100%. Such identity as compared to a reference sequence can be determined using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0053] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0054] A comparison window includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0055] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al.). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0056] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0057] Any of the vectors disclosed herein can be introduced into cells (using any techniques known in the art) for propagation of the vectors and / or for expression of a protein(s) encoded by the vector. Accordingly, provided herein is a recombinant cell andpopulations of cells comprising a vector disclosed herein. Further, any of the vectors described herein can be used in any method described herein or later developed for producing a rAAV, wherein the method comprises, for example, culturing the recombinant cell under conditions that result in the production of a rAAV capsid, and in some cases expression of a transgene (i.e., a gene of interest).

[0058] A variety of host cells and expression systems can be utilized to propagate any of the vectors described herein. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis), yeast (e.g., Saccharomyces Pichia); plant cell systems; insect cells, or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK293T, HEK293F, HepG2, SP210, Rl. l, B-W, L-M, BSC1, BSC40, YB / 20 and BMT10 cells).Vector Systems

[0059] Also provided is a vector system comprising at least two vectors, wherein a first vector comprises one or more helper sequences, (e.g., a helper plasmid comprising a modified E4 nucleic acid, an E2a nucleic acid, and a virus-associated (VA) RNA from an adenovirus genome). In some embodiments, the second vector comprises a transgene.

[0060] The vector system optionally comprises at least a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and / or virus-associated (VA) RNA of an adenovirus genome, wherein the at least a portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids, as compared to production using a vector system lacking the nucleic acid E4 deletion. In some embodiments, the at least a portion of nucleic acid E2a comprises a deletion.

[0061] The first vector optionally comprises one or more of a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein the at least a portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids, as compared to production using a vector system lacking the nucleic acid E4 deletion. In some embodiments, the at least a portion of nucleic acid E2a comprises a deletion. Optionally the first vector comprises the portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome.

[0062] The vector systems described herein can be used as packaging systems for preparation of a rAAV. In some embodiments, the packaging systems comprise or consistof (1) a first vector comprising a helper virus gene, e.g., a helper plasmid described herein; and (2) a second vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising an rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein. In some embodiments, the vector system comprises or consists of (1) a first vector that comprises one or more helper virus genes, e.g., a helper plasmid described herein; (2) a second vector that comprises the rAAV genome comprising a transgene; and (3) a third vector comprising a nucleotide sequence encoding an AAV Rep protein and a nucleotide sequence encoding an AAV capsid protein.

[0063] In some embodiments, the two or more vectors of the packaging system, together, are capable of providing all the components needed for the production of rAAV. In certain embodiments, certain components required for the production of rAAV are provided by the host cell from which rAAV are produced. In such an embodiment, the two or more vectors of the packaging system together with the host cell, are capable of providing all the components needed for the production of rAAV. The packaging systems described herein are operative in a cell for enclosing the AAV genome in a capsid to form the rAAV.

[0064] In some vector systems, the first vector comprises at least a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein the portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids as compared to production using a vector lacking the nucleic acid E4 deletion.

[0065] In some embodiments, the nucleic acid E4 deletion is a deletion of orf 1-3, resulting in a nucleic acid E4 comprising E4orf4, Eorf6 and Eorf7. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, or 1075 base pairs) of a nucleic acid sequence comprising E4orfl-E4orf3 (SEQ ID NO: 2). In some embodiments, the nucleic acid E4 comprising a deletion of E4orfsl-3 comprises a nucleic acid sequence comprising SEQ ID NO: 1 or a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 1.

[0066] In some embodiments, the vector comprises orf6-7 of nucleic acid E4; nucleic acidE2a or a portion thereof; and VA RNA of an adenoviral genome, wherein nucleic acid E4 comprises a deletion. In some embodiments, the nucleic acid E4 comprising orf6-7 alsocomprises a deletion of one or more orfs selected from the group consisting of E4orfl, E4orf2, and E4orf4. In some embodiments, the nucleic acid E4 deletion is a deletion of at least 1000 base pairs (e.g., at least about 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350 or 1400 base pairs) of a nucleic acid sequence comprising E4orfl-E4orf4 (SEQ ID NO: 5). An exemplary nucleotide sequence comprising a nucleic acid comprising E4orf6, a nucleic acid comprising E4orf7, and a nucleic acid E4 deletion of E4orfsl-4 is set forth herein as SEQ ID NO: 4. In some embodiments, the nucleic acid E4 comprising a nucleic acid comprising E4orf6, a nucleic acid comprising E4orf7, and a nucleic acid E4 deletion of E4orfsl-4 has at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 4.

[0067] In some embodiments, the at least a portion of nucleic acid E2a comprises a deletion, for example, a deletion of at least about 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 base pairs in an untranslated genomic region of the nucleic acid E2a, for example, a 5’ untranslated region (UTR). Exemplary nucleic acid sequences comprising a nucleic acid E2a comprising a deletion of at least 500 base pairs in the 5’ UTR are set forth herein as SEQ ID NOs: 8- 12. SEQ ID NO: 8 is a nucleic acid E2a comprising about a 2 kb deletion in the 5’ UTR. SEQ ID NO: 9 is a nucleic acid E2a comprising about a 1 kb deletion in the 5’ UTR. SEQ ID NO: 10 is a nucleic acid E2a comprising about a 1.4 kb deletion in the 5’ UTR. SEQ ID NO: 11 is a nucleic acid E2a comprising about a 500 base pair deletion in the 5’ UTR. SEQ ID NO: 12 is a nucleic acid E2a comprising about a 900 base pair deletion in the 5’ UTR.

[0068] In some embodiments, the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both as compared to a control AAV product produced by an AAV vector system lacking deletion of nucleic acids E4 or lacking deletions of nucleic acid E4 and nucleic acid E2a. In some embodiments, the second vector comprises a transgene. As used throughout, an increase can be an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater. An increase can also be a fold increase, for example at least a 2-4 fold increase. For example, when a control VG / L value is about 2 X 1014, the AAV product produced using the vector system described herein can be about 3-8 X 1014, e.g., 3-7 X 1014 VG / L. Similarly, when the control capsids / L is 0.75 X 1014, the AAV product producedusing the vector system described herein can be about 1-5 X 1015, e.g., 1-3 X 1015 capsids / liter.Transgenes

[0069] In some embodiments, the transgene comprises one or more sequences encoding an RNA molecule. Suitable RNA molecules include, without limitation, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomirs, miRNA sponges, RNA aptazymes, RNA aptamers, mRNA, IncRNAs, ribozymes, and synthetic RNAs known in the art.

[0070] In some embodiments, the transgene encodes one or more polypeptides, or a fragment thereof. Such transgenes can comprise the complete coding sequence of a polypeptide or only a fragment of a coding sequence of a polypeptide. In certain embodiments, the transgene encodes a polypeptide that is useful to treat a disease or disorder in a subject. Suitable polypeptides include, without limitation, P-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P), and the like; soluble receptors, such as soluble TNF-a receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble y / A T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as a-glucosidase, imiglucerase, P-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, monokine induced by interferon-gamma (Mig), Groa / IL-8, RANTES, MIP-la, MIP-ip, MCP-1, PF -4, and the like; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2; and the like; anti- angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, betaendorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha- 1 antitrypsin; leukemia inhibitory factor (LIF); tissue factors; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonists; ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophins 3 and 4 / 5 (NT-3 and -4 / 5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); Factor VIII, Factor IX, Factor X; dystrophin or mini -dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6- phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, P-enolase, glycogen synthase; lysosomal enzymes, such as iduronate-2-sulfatase (I2S), and arylsulfatase A; and mitochondrial proteins, such as frataxin.

[0071] In some embodiments, the transgene encodes a protein that may be defective in one or more lysosomal storage diseases. Suitable proteins include, without limitation, a- sialidase, cathepsin A, a-mannosidase, P-mannosidase, glycosylasparaginase, a- fucosidase, a-N-acetylglucosaminidase, P-galactosidase, P-hexosaminidase a-subunit, P- hexosaminidase P-subunit, GM2 activator protein, glucocerebrosidase, Saposin C, Arylsulfatase A, Saposin B, formyl -glycine generating enzyme, P-galactosylceramidase, a-galactosidase A, iduronate sulfatase, a-iduronidase, heparan N-sulfatase, acetyl-CoA transferase, N-acetyl glucosaminidase, P -glucuronidase, N-acetyl glucosamine 6- sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, a- glucosidase, acid sphingomyelinase, acid ceramidase, acid lipase, cathepsin K, tripeptidyl peptidase, palmitoyl -protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine, phosphotransferase y-subunit, mucolipin-1, LAMP -2, NPC1, CLN3, CLN 6, CLN 8, LYST, MYOV, RAB27A, melanophilin, and AP3 P-subunit.

[0072] In some embodiments, the transgene encodes an antibody or a fragment thereof (e.g., a Fab, scFv, or full-length antibody). Suitable antibodies include, without limitation,muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin, durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cemiplimab, fremanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab.

[0073] In some embodiments, the transgene encodes a nuclease. Suitable nucleases include, without limitation, zinc fingers nucleases (ZFN) (see, e.g., Porteus, and Baltimore (2003) Science 300: 763; Miller et al. (2007) Nat. Biotechnol. 25:778-785; Sander et al. (2011) Nature Methods 8:67-69; and Wood et al. (2011) Science 333:307, each of which is hereby incorporated by reference in its entirety), transcription activator-like effectors nucleases (TALEN) (see, e.g., Wood et al. (2011) Science 333:307; Boch et al. (2009) Science 326: 1509-1512; Moscou and Bogdanove (2009) Science 326: 1501; Christian et al. (2010) Genetics 186:757-761; Miller et al. (2011) Nat. Biotechnol. 29: 143-148; Zhang et al. (2011) Nat. Biotechnol. 29: 149-153; and Reyon et al. (2012) Nat. Biotechnol. 30(5): 460-465, each of which is hereby incorporated by reference in its entirety), homing endonucleases, meganucleases (see, e.g., U.S. Patent Publication No. US 2014 / 0121115, which is hereby incorporated by reference in its entirety), and RNA-guided nucleases (see, e.g., Makarova et al. (2018) The CRISPR Journal 1(5): 325-336; and Adli (2018) Nat. Communications 9: 1911, each of which is hereby incorporated by reference in its entirety).

[0074] In some embodiments, the transgene encodes an RNA-guided nuclease. Suitable RNA-guided nucleases include, without limitation, Class I and Class II clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases. Class I isdivided into types I, III, and IV, and includes, without limitation, type I (Cas3), type I-A (Cas8a, Cas5), type I-B (Cas8b), type I-C (Cas8c), type I-D (CaslOd), type I-E (Csel, Cse2), type I-F (Csyl, Csy2, Csy3), type I-U (GSU0054), type III (CaslO), type III-A (Csm2), type III-B (Cmr5), type III-C (CsxlO or Csxl 1), type III-D (CsxlO), and type IV (Csfl). Class II is divided into types II, V, and VI, and includes, without limitation, type II (Cas9), type II-A (Csn2), type II-B (Cas4), type V (Cpfl, C2cl, C2c3), and type VI (Casl3a, Casl3b, Casl3c). RNA-guided nucleases also include naturally-occurring Class II CRISPR nucleases such as Cas9 (Type II) or Casl2a / Cpfl (Type V), as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).

[0075] In some embodiments, the transgene encodes one or more reporter sequences, which upon expression produce a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding P-lactamase, P -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein (RFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins, including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.AAV genomes

[0076] In some embodiments, the rAAV genome comprises a transcriptional regulatory element (TRE) operably linked to the transgene to control expression of an RNA or polypeptide encoded by the transgene. In certain embodiments, the TRE comprises a constitutive promoter. In certain embodiments, the TRE can be active in any mammalian cell (e.g., any human cell). In certain embodiments, the TRE is active in a broad range of human cells. Such TREs may comprise constitutive promoter and / or enhancer elements, including any of those described herein. In certain embodiments, the TRE comprises an inducible promoter. In certain embodiments, the TRE may be a tissue-specific TRE, i.e., it is active in specific tissue(s) and / or organ(s). A tissue-specific TRE comprises one or more tissue-specific promoters and / or enhancer elements, and optionally one or more constitutive promoters and / or enhancer elements. A skilled artisan would appreciate thattissue-specific promoters and / or enhancer elements can be isolated from genes specifically expressed in the tissue by methods well known in the art.

[0077] Suitable promoters include, for example, and not to be limiting, cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441), CMV early enhancer / chicken P-actin (CBA) promoter / rabbit P-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2): 269-277), CBSB (Jacobson et al. (2006) Molecular Therapy 13(6): 1074-1084), human elongation factor la promoter (EFla) (Kim et al. (1990) Gene 91 (2): 217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417), mitochondrial heavy-strand promoter (Lodeiro et al. (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261 : 101- 105). In certain embodiments, the TRE is brain-specific (e.g., neuron-specific, glial cellspecific, astrocyte-specific, oligodendrocyte-specific, microglia-specific and / or central nervous system-specific). Exemplary brain-specific TREs may comprise one or more elements from, without limitation, human glial fibrillary acidic protein (GFAP) promoter, human synapsin 1 (SYN1) promoter, human synapsin 2 (SYN2) promoter, human metallothionein 3 (MT3) promoter, and / or human proteolipid protein 1 (PLP1) promoter. More brain-specific promoter elements are disclosed in WO 2016 / 100575A1, which is incorporated by reference herein in its entirety.

[0078] In some embodiments, the native promoter for the transgene may be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0079] In certain embodiments, the rAAV genome comprises an editing genome. As used herein, the term editing genome refers to a recombinant AAV genome that is capable of modifying a genomic target locus via homologous recombination. Editing genomes can be used to edit the genome of a cell by homologous recombination of the editing genome with a genomic region surrounding a target locus in the cell. In certain embodiments, the editing genome is designed to correct a genetic defect in a gene by homologous recombination. Editing genomes generally comprise (i) an editing element for editing or modifying a target locus in a target gene; (ii) a 5' homology arm nucleotide sequence 5' ofthe editing element having homology to a first genomic region 5' to the target locus; and (iii) a 3' homology arm nucleotide sequence 3' of the editing element having homology to a second genomic region 3' to the target locus, wherein the portion of the editing genome comprising the 5' homology arm, editing element, and 3' homology arm can be in the sense or antisense orientation relative to the target locus. Suitable target genes for editing using an editing genome include, without limitation, phenylalanine hydroxylase (PAH), cystic fibrosis conductance transmembrane regulator (CFTR), beta hemoglobin (HBB), oculocutaneous albinism II (0CA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y).

[0080] In some embodiments, the rAAV genomes disclosed herein further comprise a transcription terminator (e.g., a polyadenylation sequence). In certain embodiments, the transcription terminator is 3' to the transgene. The transcription terminator may be any sequence that effectively terminates transcription, and a skilled artisan would appreciate that such sequences can be isolated from any genes that are expressed in the cell in which transcription of the at least a portion of an antibody coding sequence is desired. In certain embodiments, the transcription terminator comprises a polyadenylation sequence. In certain embodiments, the polyadenylation sequence is identical or substantially identical to the endogenous polyadenylation sequence of an immunoglobulin gene. In certain embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence. In certain embodiments, the polyadenylation sequence is an SV40 polyadenylation sequence. In certain embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence.

[0081] In certain embodiments, the rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence 5' of the TRE, and a 3' inverted terminal repeat (3' ITR) nucleotide sequence 3' of the polyadenylation sequence. ITR sequences from any AAV serotype or variant thereof can be used in the rAAV genomes disclosed herein. The 5' and 3' ITR can be from an AAV of the same serotype or from AAVs of different serotypes.

[0082] In certain embodiments, the 5' ITR or 3' ITR is from AAV2. In certain embodiments, the 5' ITR or 3' ITR are from AAV5. In certain embodiments, both the 5' ITR and 3' ITR are from AAV5 or AAV2.

[0083] In certain embodiments, the 5' ITR nucleotide sequence and the 3' ITR nucleotide sequence are substantially complementary to each other (e.g., are complementary to each other except for mismatch at 1, 2, 3, 4, or 5 nucleotide positions in the 5' or 3' ITR).

[0084] In certain embodiments, the 5' ITR or the 3' ITR is modified to reduce or abolish resolution by Rep protein (“non-resolvable ITR”). In certain embodiments, the non- resolvable ITR comprises an insertion, deletion, or substitution in the nucleotide sequence of the terminal resolution site. Such modification allows formation of a self- complementary, double-stranded DNA genome of the AAV after the rAAV genome is replicated in an infected cell. Exemplary non-resolvable ITR sequences are known in the art (see, e.g., those provided in U.S. Patent Nos. 7,790,154 and 9,783,824, which are incorporated by reference herein in their entirety).Rep protein

[0085] In the vector systems described herein, expression of the AAV Rep gene is controlled through the use of two promoters and alternative splicing, and results in four Rep proteins, Rep78, Rep68, Rep52, and Rep40. The Rep proteins are involved in AAV genome replication and packaging of the viral genome. Expression of Rep proteins is controlled by the p5 and pl9 promoters. The p5 promoter drives expression of the alternative splice variants Rep78 and Rep68. The pl9 promoter drives expression of the alternative splice variants Rep52 and Rep40. Accordingly, the vector systems provided herein can include a vector comprising a nucleotide sequence encoding one or more Rep proteins or functional variants thereof.

[0086] The one or more Rep proteins may be derived from AAV2. An exemplary AAV2 genome sequence can be found via NCBI Reference Sequence NC_001401.2. According to the NCBI Reference Sequence, Rep68 is encoded by nucleotides 321 to 2252; Rep78 is encoded by nucleotides 321 to 2186; Rep40 is encoded by nucleotides 993 to 2252; and Rep52 is encoded by nucleotides 993 to 2186. In certain embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence corresponding to the sequence encoding Rep40, Rep68, Rep78, Rep52 as described for AAV2, in a different adenovirus serotype, for example, AAV5.AAV capsid

[0087] The vector systems provided herein comprise a nucleic acid vector comprising a nucleotide sequence encoding an AAV capsid protein coding sequence. The nucleotide sequence can encode an AAV capsid protein from any AAV capsid known in the art, including natural AAV isolates and variants thereof.

[0088] AAV capsid proteins include VP1, VP2, and VP3 capsid proteins. VP1, VP2, and / or VP3 capsid proteins assemble into a capsid that surrounds the rAAV genome. In certain embodiments, assembly of the capsid proteins is facilitated by the assembly-activating protein (AAP). Capsids of certain AAV serotypes require the role of AAP in transporting the capsid proteins to the nucleolus for assembly. For example, AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV12 require AAP to form capsids, while capsids of AAV4, AAV5, and AAV11 can assemble without AAP. See, e.g., Earley et al. (2017) J. Virol. 91(3): e01980-16.

[0089] Different AAV serotypes or variants thereof comprise AAV capsid proteins having different amino acid sequences. Suitable AAV capsid proteins include, without limitation, a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, AAVrhlO, AAVRh32.33, AAVrh74, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, AAVHSC17, and any variants thereof. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhlO and AAVrh74. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8 and AAVrh74. The sequences of the various AAV capsid proteins are disclosed in, e.g., U.S. Patent Publication Nos. US20030138772, US20140359799, US20150159173, US20150376607, US20170081680, and US20170360962A1, and PCT Publication No. WO2020227515, the disclosures of which are incorporated by reference herein in their entireties. rAAV Production

[0090] Also provided is a method of producing an AAV product comprising an AAV capsid. The method comprises (a) introducing into a host cell any of the vector systemsdescribed herein, (b) incubating the host cells under culture conditions that promote AAV product production, and (c) purifying the AAV product.

[0091] In some embodiments, the host cell is a mammalian cell, for example, a mammalian cell selected from the group consisting of a COS cell, a CHO cell, a BHK cell, an MDCK cell, an HEK293 cell, an HEK293T cell, a HeLa cells, an NSO cell, a PER.C6 cell, a VERO cell, a CRL7O3O cell, an HsS78Bst cell, a HeLa cell, an NIH 3T3 cell, a HepG2 cell, an SP210 cell, an Rl.l cell, a B-W cell, an L-M cell, a BSCl cell, a BSC40 cell, a YB / 20 cell, and a BMT10 cell. Optionally, the host cell is a cell that can be grown in suspension culture, for example, an HEK293 cell, an HEK293T, or an HEK293F cell.

[0092] In some embodiments, rAAV production comprises an upstream production process that generally comprises expanding mammalian cells to an appropriate cell density, introducing one or more vectors into the expanded cells to generate AAV producer cells, culturing the AAV producer cells under conditions to produce rAAV particles (capsids), and harvesting and lysing the AAV producer cells for subsequent recovery of the rAAV particles. After recovery of the rAAV particles, downstream production processes ensure the sufficient purification of the rAAV particles from contaminants.

[0093] As used herein, the term introducing (i.e., transfecting or transducing), in the context of introducing a nucleic acid sequence, for example, one or more vectors described herein, refers to the translocation of the nucleic acid sequence from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like.

[0094] In some embodiments, the vectors introduced into the expanded cells include a first vector comprising one or more helper virus genes, e.g., a helper plasmid described herein and a second vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising an rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein. When the first vector comprising one or more helper virus genes and the second vector comprising nucleotide sequences encoding AAV Rep protein, a transgene, and an AAV capsid protein are introduced into the expanded cells, this can occur at various molar ratios most appropriate for the vectors in use, for example an equal molar 1 : 1 ratio. In someembodiments, the vectors introduced into the expanded cells include a first vector comprising one or more helper virus genes, e.g., a helper plasmid described herein; a second vector comprising the rAAV genome comprising a transgene; and a third vector comprising a nucleotide sequence encoding an AAV Rep protein, and a nucleotide sequence encoding an AAV capsid protein. When the first vector comprising one or more helper virus genes, the second vector comprising a transgene, and third vector comprising an AAV capsid protein and an AAV capsid protein are introduced into the expanded cells, this can occur at various molar ratios most appropriate for the vectors in use, for example at a 1 :2:2 ratio. The total amount of nucleic acid that is introduced into the cell is from about 0.1 pg DNA / 1 X 106 cells to 4.0 pg DNA / 1 X 106 cells. For example, the total amount of nucleic acid that is transfected or transduced into the cell, including the first nucleic acid vector, the second nucleic acid vector, and optionally the third nucleic acid vector is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 pg DNA / 1 X 106 cells.

[0095] In some embodiments, the mammalian cell is provided in a cell culture. In certain embodiments, the cell culture has a volume of at least 2-5000 liters. For example, at least 50 liters, at least 500 liters, or at least 2000 liters. In certain embodiments, the methods described herein are carried out in a bioreactor having a volume of at least 2 liters, at least 50 liters, or at least 2000 liters.

[0096] Methods for purifying an AAV product are known in the art. As used herein, purify or purification refers to the elimination of at least a portion of a non- AAV product, e.g., removal of host cell proteins, media components, nucleic acids, and / or empty AAV capsids from a cell culture, to obtain a purified AAV product. For example, in some methods at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 99.5% or 100% of one or more non-AAV products can be removed or eliminated during purification. In some cases, purification comprises (a) a clarification step for the removal of cells and cellular debris, e.g., using differential centrifugation, density centrifugation and / or filtration; and / or (b) one or more downstream chromatography steps to separate the AAV product from various impurities in the clarified cell culture feed. See, e.g., International Patent Application Publication No. WO 98 / 22588, which describes methods for the production and purification of adenoviral vectors. Chromatographic methods for purification of virus from a host celllysate, are also set forth in U.S. Pat. Nos. 6,008,036, 6,586,226, 5,837,520, 6,261,823, 6,537,793, and International Patent Application Publication Nos. WO 00 / 50573, WO 02 / 44348 and WO 03 / 078592, the contents of the entirety of each of which are incorporated herein by this reference. Various chromatographic and non-chromatographic methods can be used, including affinity chromatography, anion exchange chromatography, ultracentrifugation, and other methods in the art.Methods of Use

[0097] Provided is a method of delivering a transgene to a cell, comprising introducing into the cell an AAV product produced by the methods described herein. Introduction into a cell is typically by way of infection.

[0098] Also provided is a method of treating a subject in need of a protein produced by a transgene, comprising administering to the subject an effective amount of an AAV product produced by any of the methods of producing an AAV product described herein.

[0099] In some embodiments, the AAV product is an AAV particle or virion. As used herein, a recombinant AAV particle or virion is a viral particle including at least one AAV capsid protein and an encapsidated recombinant AAV viral genome comprising a transgene. The transgene delivered to a cell, optionally in a subject, can be heterologous to the cell. As used herein, “heterologous” refers to what is not normally found in nature. As such, a heterologous nucleotide sequence may be (a) foreign to its host cell (i.e., is exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.

[0100] The term effective amount, as used throughout, is defined as any amount necessary to produce a desired physiologic response, for example, reducing or delaying one or more effects or symptoms of a disease or disorder. Effective amounts and schedules for administering the AAV product, for example, recombinant AAV particles, can be determined empirically and making such determinations is within the skill in the art.

[0101] The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, unwanted cell death, and the like. Generally, the dosage will vary with the species, age, body weight, general health, sexand diet of the subject, the mode and time of administration and severity of the particular condition and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more doses.

[0102] An effective amount of any of the recombinant AAV virions described herein will vary and can be determined by one of skill in the art through experimentation and / or clinical trials. For example, an effective dose can be from about 106 to about 1015 recombinant rAAV virions, or any values in between this range, for example, about 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 recombinant AAV particles. Thus, the number of rAAV particles administered to a subject may be on the order ranging from about 106 to 1015 vector genomes(vgs) / ml, such as for example, about 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 vg / ml. In some embodiments, the number of rAAV particles administered to a subject can be from about 106 to 1015 vg / kg, or any values in between these amounts, such as for example, about 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 vg / kg. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0103] The compositions described herein are administered in a number of ways depending on whether local or systemic treatment is desired. The compositions are administered via any of several routes of administration, including intraparenchymal injection, intravenously, intrathecally, intramuscularly, intraci stemally, intracoronary injection, intramyocardium injection, intradermally, endomyocardiac injection, or a combination thereof. Effective doses for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0104] As used throughout, patient is used interchangeably with subject. By subject is meant an individual. The subject can be an adult subject or a pediatric subject. Pediatric subjects include subjects ranging in age from birth to eighteen years of age. Preferably, the subject is an animal, for example, a mammal such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

[0105] As used throughout, treat, treating, and treatment refer to a method of reducing or delaying one or more effects or symptoms of a disease or disorder. The subject can be diagnosed with the disease or disorder. Treatment can also refer to a method of reducing the underlying pathology rather than just the symptoms. The effect of the administration to the subject can have the effect of, but is not limited to, reducing one or more symptoms of the disease, a reduction in the severity of the disease, the complete ablation of the disease, or a delay in the onset or in the worsening of one or more symptoms. For example, a disclosed method is considered to be a treatment if there is about a 10% reduction in one or more symptoms of the disease when compared to the subject prior to treatment or when compared to a control subject or control value. Thus, the reduction can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.Embodiments1. A vector comprising nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein nucleic acid E4 comprises a deletion of open reading frames 1-3.2. The vector of embodiment 1, wherein the nucleic acid E4 deletion is a deletion of at least 1000 base pairs.3. The vector of embodiment 1 or 2, wherein the nucleic acid E4 deletion is a deletion of at least 1000 base pairs of E4orfl-3.4. The vector of any one of embodiments 1-3, wherein the nucleic acid E4 deletion is a deletion of at least 1400 base pairs of E4orfl-4.5. The vector of any one of embodiments 1-4, wherein nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4; or a nucleic acid sequence having at last 85% identity to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4.6. The vector of any one of embodiments 1-5, wherein nucleic acid E4 comprises open reading frame 6.7. The vector of embodiment 6, wherein the nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid sequence having at last 85% identity to SEQ ID NO:3.8. The vector of embodiment 6 or 7, wherein nucleic acid E4 further comprises open reading frame 7.9. The vector of embodiment 8, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:4 or a nucleic acid sequence having at last 85% identity to SEQ ID NO:4.10. The vector of any one of embodiments 1-9, wherein the vector further comprises a nucleic acid E2a deletion.11. The vector of embodiment 10, wherein the vector comprises open reading frame 6 (orf6) of nucleic acid E4, the nucleic acid E2a deletion, and VA RNA of an adenoviral genome.12. The vector of embodiment 10, wherein the vector comprises open reading frame 6 (orf6) of nucleic acid E4, open reading frame 7 (orf7) of nucleic acid E4, the nucleic acid E2a deletion; and VA RNA of an adenoviral genome.13. The vector of any one of embodiments 10-12, wherein the E2 deletion comprises a deletion of greater than 800 base pairs in the 5’ untranslated region of the E2a gene.14. The vector of embodiment 13, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.15. The vector of any one of embodiments 1-14, wherein the vector comprises a nucleic acid sequence of SEQ ID NO: 13, 14,15, 16, 17, or 18.16. A vector system comprising at least two vectors, wherein a first vector comprises a helper plasmid, wherein the helper plasmid is selected from the vector of any one of embodiments 1-15.17. The vector system of embodiment 16, wherein a second vector comprises a transgene.18. A vector system comprising at least two vectors, wherein a first vector comprises at least a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein the at least a portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids as compared to production using a vector lacking the nucleic acid E4 deletion.19. The vector system of embodiment 18, wherein at least a portion of nucleic acid E2a comprises a deletion.20. The vector system of embodiment 18 or 19, wherein the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both as compared toa control AAV product produced by an AAV vector system lacking deletion of nucleic acids E4 or lacking deletions of nucleic acid E4 and nucleic acid E2a.21. The vector system of any one of embodiments 18-20, wherein a second vector comprises a transgene.22. A method of producing an AAV product comprising an AAV capsid, comprising(a) introducing into a host cell the vector system of any one of embodiments 18-21,(b) incubating the host cells under culture conditions that promote AAV product production, and(c) purifying the AAV product.23. A method of treating a subject in need of a protein produced by transgene, comprising administering to the subject an AAV product produced by the method of embodiment 22.24. A vector system comprising one or more vectors which together comprise nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein nucleic acid E4 comprises a deletion of open reading frames 1-3.25. The vector system of embodiment 24, wherein nucleic acid E4 comprises a deletion of open reading frames 1-4.26. The vector system of embodiments 23-25 wherein the vector further comprises an E2a deletion.EXAMPLES

[0106] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

[0107] Helper plasmids for AAV production can be challenging to produce at large scale with high density fermentation, resulting in low yields and increased manufacturing costs. FIG. l is a schematic of a traditional helper plasmid construct, including VA, E4, and E2a adenovirus sequences. Therefore, in an effort to improve the manufacturability of the plasmid, helper plasmid size was reduced by removing regions of the adenoviral genes that were not essential to their function. Five helper plasmid (pHelpers) deletion constructs aimed at reducing the size of E4 and E2a transcriptional units, as well as reducing the plasmid backbone, were compared with a control plasmid that does notcomprise the test deletions (control pHelper). See FIG. 2. Surprisingly, all five helper plasmids that included a deletion of E4 (without or without additional deletions of E2a) increased vector genome (VG) titers across multiple genomes. See. FIG. 3. Capsid production also increased proportionally to VGs using the helper plasmids with E4 deletions (without or without the deletions of E2a). See FIG. 3. It is likely that removal of non-coding regions of the adenoviral genes alters the expression of E2a and E4 proteins, which enables higher capsid expression and in turn increased the number of VGs packaged into the available capsids. Productivity was optimized in the construct that included deletion of orfl-4 of E4, deletions of E2a as described herein, and backbone deletions. See. FIG. 4.Example 1: Dual Plasmid Transfection

[0108] Host cells were expanded for at least one passage, inoculated into a flask containing the appropriate amount of cell culture medium, and incubated in a shaking incubator at 37°C, with 8% CO2, and 135 rpm. Cells were transfected when they reached a density of 2E6 cells / mL. For dual plasmid transfection, a total of 0.75ug DNA / 1 X 106 was transfected into host cells. The transfection plasmids include a first vector comprised of a GFP -Luciferase transgene and an AAV2 Rep gene and an AAV9 Cap gene, and a second vector comprised of either control or various pHelper plasmid constructs (Del 1-5) (Table 1). Transfection plasmids were combined at an equal molar ratio of 1 : 1. Transfection mixes were prepared by mixing calculated volumes of vector(s) and polyethylenimine (PEI), all at ambient temperature at a molar ratio of 1.5: 1 of PEI to DNA (Table 1). The transfection mixes were then added into the flasks and incubated in a shaker at 37°C, with 8% CO2, and 135 rpm, for 72 hours before harvesting. After 72 hours of incubation, cells were lysed using a lysis buffer containing IM Tris (pH 9.5), 10% Triton X-100, IM MgC12, endonuclease (e.g., BENZONASE®, DENARASE®), and 5M NaCl, and the shake flasks were incubated for 60 minutes at 37°C, with 8% CO2, and 135 rpm. Crude lysate samples were collected by centrifugation.Table 1: Plasmid TransfectionPlasmid ratios are molar based.Example 2: Determination of Vector Genome and Capsid Productivity from DualPlasmid Transfection

[0109] Vector genome titer (vg / L) was determined by droplet digital PCR (ddPCR) by standard methods using primer / probe sets specific to the transgene payload of the vector comprising the transgene (i.e., transgene vector) (FIG. 3). The number of capsids per cell was determined using enzyme-linked immunosorbent assays (ELISAs) by standard methods with an immobilized antibody directed against an epitope of the capsid as encoded by the vector comprising the Cap sequences (FIG. 3).Example 3: Triple Plasmid Transfection

[0110] Host cells were expanded for at least one passage, inoculated into a flask containing the appropriate amount of cell culture medium, and incubated in a shaking incubator at 37°C, with 8% CO2, and 135 rpm. Cells were transfected when they reached a density of 1 X 106 cells / mL. For triple plasmid transfection, a total of 0.75ug DNA / 1 X 106 was transfected into host cells. The transfection plasmids include a first vector comprised of a GFP -Luciferase transgene, a second vector comprised of Rep / Cap proteins across multiple AAV serotype backgrounds including HSC15, AAV9, and AAV2, and a third vector comprised of Del 5 (Table 2). Transfection plasmids were combined at an equal ratio of 1 :2:2 (GFP-Luciferase:Rep / Cap:pHelper). Transfection mixes were prepared by mixing calculated volumes of vector(s) and polyethylenimine (PEI), all at ambient temperature at a ratio of 1.5: 1 of PEI to DNA (Table 2). The transfection mixes were then added into the flasks and incubated in a shaker at 37°C, with 8% CO2, and 135 rpm, for 72 hours before harvesting. After 72 hours of incubation, cells were lysed using a lysis buffer containing IM Tris (pH 9.5), 10% Triton X-100, IM MgC12, endonuclease (e.g., BENZONASE®, DENARASE®), and 5M NaCl, and the shake flasks were incubated for 60 minutes at 37°C, with 8% CO2, and 135 rpm. Crude lysate samples were collected by centrifugation.Table 2: Triple Plasmid TransfectionPlasmid ratios are mass based.Example 4 Determination of Vector Genome and Capsid Productivity from Triple Plasmid Transfection

[0111] Vector genome titer (vg / L) was determined by droplet digital PCR (ddPCR) by standard methods using primer / probe sets specific to the transgene payload of the vector comprising the transgene (i.e., transgene vector) (FIG. 4). The number of capsids per cell was determined using enzyme-linked immunosorbent assays (ELISAs) by standard methods with an immobilized antibody directed against an epitope of the capsid as encoded by the vector comprising the Cap sequences (FIG. 4).

Claims

CLAIMS1. A vector comprising nucleic acid E4, nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein nucleic acid E4 comprises a deletion of open reading frames 1 -3.

2. The vector of claim 1, wherein the nucleic acid E4 deletion is a deletion of at least 1000 base pairs.

3. The vector of claim 2, wherein nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4; or a nucleic acid sequence having at last 85% identity to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4.

4. The vector of claim 1, wherein nucleic acid E4 comprises open reading frame 6.

5. The vector of claim 4, wherein the nucleic acid E4 comprises the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid sequence having at last 85% identity to SEQ ID NO:3.

6. The vector of claim 4, wherein nucleic acid E4 further comprises open reading frame 7.

7. The vector of claim 6, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:4 or a nucleic acid sequence having at last 85% identity to SEQ ID NO:4.

8. The vector of claim 1, wherein the vector further comprises a nucleic acid E2a deletion.

9. The vector of claim 8, wherein the E2 deletion comprises a deletion of greater than 800 base pairs in the 5’ untranslated region of the E2a gene.

10. The vector of claim 9, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

11. The vector of claim 1, wherein the vector comprises a nucleic acid sequence of SEQ ID NO: 13, 14,15, 16, 17, or 18.

12. A vector system comprising a first vector and a second vector, wherein the first vector is the vector of claim 1.

13. The vector system of claim 12, wherein the second vector comprises a transgene.

14. A vector system comprising a first vector and a second vector, wherein the first vector comprises at least a portion of nucleic acid E4, at least a portion of nucleic acid E2a, and virus-associated (VA) RNA of an adenovirus genome, wherein the at least a portion of nucleic acid E4 comprises a deletion configured to increase production of an AAV product comprising AAV capsids as compared to production using a vector lacking the nucleic acid E4 deletion.

15. The vector system of claim 14, wherein at least a portion of nucleic acid E2a comprises a deletion.

16. The vector system of claim 14, wherein the AAV product has a higher level of vector genome per liter, a greater number of capsids per liter, or both, as compared to a control AAV product produced by an AAV vector system lacking deletion of nucleic acids E4 or lacking deletions of nucleic acid E4 and nucleic acid E2a.

17. The vector system of claim 14, wherein the second vector comprises a transgene.

18. A method of producing an AAV product comprising an AAV capsid, comprising:(a) introducing into a host cell the vector system of claim 14,(b) incubating the host cells under culture conditions that promote AAV product production, and(c) purifying the AAV product.

19. A method of delivering a transgene to a cell, the method comprising introducing into the cell the AAV product produced by the method of claim 18.

20. The method of claim 19, which is an in vitro method.

21. The method of claim 19, wherein the transgene is expressed by the cell.

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