Adeno-associated virus (AAV) production

Abstract

The present disclosure relates to methods for producing adeno-associated virus (AAV) in E1-complementing producer cells.

Description

[Technical field]

[0001] The present disclosure relates to methods for producing adeno-associated virus (AAV) in E1-complementing producer cells. [Background technology]

[0002] Adeno-associated virus (AAV) is a non-enveloped DNA virus. As the primary viral vector for in vivo gene therapy, AAV is non-pathogenic and can effectively infect a wide range of human tissues. The AAV genome contains 4.7 kb of single-stranded DNA encoding two major genes: the Rep gene encodes four isoforms (Rep78, Rep68, Rep52, and Rep40) that support AAV DNA replication, packaging, integration, and transcription, and the Cap gene synthesizes three VP proteins (VP1, VP2, and VP3) for viral protein shell or capsid assembly.

[0003] Wild-type AAV cannot replicate without helper viruses, such as adenovirus (Ad) and herpes simplex virus (HSV). Adenovirus helper genes have been identified as necessary and sufficient for AAV replication, including E1A, E1B, E2A, E4, and VA. Adenovirus type 5 (Ad5) 5'4.3 kb genomic DNA is integrated into chromosome 19 of human embryonic kidney 293 (HEK293) cells. This Ad5 genomic fragment expresses all E1A (e.g., 289R, 243R, 171R) and E1B (e.g., 19K and 55K) isoforms. Additional cell lines with E1A and E1B integration have been developed, including, for example, PER.C6 cells and CAP® cells. See, e.g., Fallaux et al., Human Gene Ther 9(13):1909-1917 (1998) and Schiedner et al., Human Gene Ther 11(15):2105-2116 (2000). Thus, these cells already express E1A and E1B and can therefore support recombinant AAV (rAAV) production by transfecting a "triple plasmid transfection" approach, i.e., a helper plasmid (pHelper) encoding only E2A, E4orf6, and VA, together with a plasmid encoding the Rep and Cap genes (pRepCap) and the gene of interest (pAAV-GOI). Such cells, which have genomically integrated E1A and E1B genes and naturally express E1A and E1B proteins, are known as "E1-complementing producer cells."

[0004] Currently, over 70% of the AAV-based gene therapy pipeline in the world relies on a manufacturing platform via transient transfection in HEK293 cells with a standard triple plasmid transfection method. The productivity of HEK293 and other E1-complementing producer cell manufacturing platforms may not be able to meet the growing demand for AAV, for example, for clinical development and commercialization. However, current approaches to increase AAV productivity in E1-complementing producer cells such as HEK293 cells, such as high-producer cell clone screening and isolation, transfection protocol optimization, and manufacturing process development, are costly and time-consuming. Summary of the Invention

[0005] In some embodiments, the disclosure provides methods of producing adeno-associated virus (AAV) in E1-complementing producer cells, comprising: (a) transfecting an E1-complementing producer cell with one or more vectors comprising: (i) an E1A adenoviral helper gene; (ii) an adenoviral helper gene selected from E2A, E4, or both; (iii) a virus-associated, non-coding RNA (VA RNA); and (iv) an AAV gene selected from Rep, Cap, or both; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV.

[0006] In additional embodiments, the disclosure provides a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A operably linked to a first promoter, (ii) a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter, and (iii) a third vector comprising Rep and Cap, both operably linked to a third promoter, wherein the transfection ratio of (i) to each of (ii) and (iii) is from about 1:1 to about 3:1; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV. Suitably, one or more of the first, second or third vector further comprises a helper gene selected from E1B, NP1, NS2, or a combination thereof. Suitably, E1B is E1B-19k or E1B-55k. Suitably, the helper gene comprises a combination of NP1 and NS2. In an embodiment, the method further comprises transfecting the E1 complementation producer cell with a vector comprising the GOI.

[0007] In a further embodiment, the disclosure provides a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A operably linked to a first promoter and enhancer, (ii) a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter, and (iii) a third vector comprising Rep and Cap, each operably linked to a third promoter; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV, wherein E1A is expressed in a ratio of about 1:1 to about 3:1 relative to each of E2A, E4, VA RNA, Rep, and Cap; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV. Suitably, one or more of the first, second or third vector further comprises a helper gene selected from E1B, NP1, NS2, or a combination thereof. Suitably, E1B is E1B-19k or E1B-55k. Suitably, the helper gene comprises a combination of NP1 and NS2. In an embodiment, the method further comprises transfecting the E1 complementation producer cell with a vector comprising the GOI.

[0008] In still further embodiments, the disclosure provides a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer with (i) a first vector comprising E1A, E2A, E4, and VA RNA, all operably linked to a first promoter, and (ii) a second vector comprising Rep and Cap, operably linked to a second promoter, wherein the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and Cap is from about 1:1 to about 3:1; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV. Suitably, one or both of the first or second vectors further comprise a helper gene selected from E1B, NP1, NS2, or a combination thereof. Suitably, E1B is E1B-19k or E1B-55k. Suitably, the helper genes comprise a combination of NP1 and NS2. In an embodiment, the method further comprises transfecting the E1 complementation producer cell with a vector comprising the GOI. [Brief description of the drawings]

[0009] The following drawings form part of the present specification and are included to further demonstrate illustrative embodiments of certain aspects of the present invention.

[0010] [Figure 1] As described in the embodiments herein, standard triple transfection plasmids for AAV production in E1-complementing producer cells are shown: pHelper plasmid containing E2A, E4, and VA RNAs, pRep-Cap plasmid containing Rep and Cap, and pAAV-GOI plasmid containing inverted terminal repeat (ITR) sequences and a gene of interest (GOI). [Diagram 2]Figure 1 shows the viral titer results of the transfection experiments described in the embodiments herein. HEK293 cells were transfected with a standard triple transfection plasmid ("3xTnfx") and one or more additional helper genes (empty vector (control), E1A alone, E1B-19k alone, E1B-55k alone, NP1-NS2, E1A+E1B-19k, E1A+19B-55k, E1B-19k+E1B-55k, and E1A+E1B-19k+E1B-55k). Crude viral titers were analyzed by droplet digital PCR (ddPCR). [Figure 3A] Figure 1 shows the viral titer results of the transfection experiments described in the embodiments herein. HEK293 cells were transfected with a standard triple transfection plasmid ("3xTnfx") and one or more additional helper genes (empty vector (control), E1A alone, E1B-19k alone, E1B-55k alone, NP1-NS2, and E1A+NP1-NS2). Crude viral titers were analyzed by droplet digital PCR (ddPCR). [Figure 3B] 3 shows protein expression results from transfection experiments described in the embodiments herein. Western blot analysis was performed to evaluate protein expression levels of E1A (289R, 243R, and 171R isoforms), Rep (Rep78 and Rep52 isoforms), and Cap (VP1, VP2, and VP3) in HEK293 cells shown in FIG. 3A and table. The level of β-actin was measured as a control. [Figure 4A]Figure 1 shows protein expression results from transfection experiments described in the embodiments herein. Plasmids containing E1A were transfected at 1:1, 2:1, or 3:1 molar ratios with each of the standard triple transfection plasmids ("3xTnfx"), which are indicated as 1xE1A, 2xE1A, or 3xE1A, respectively. Western blot analysis was performed to evaluate protein expression levels of E1A (289R, 243R, and 171R isoforms), Rep (Rep78 and Rep52 isoforms), and Cap (VP1, VP2, and VP3) in HEK293 cells transfected with the plasmids indicated in the table. [Figure 4B] 1 shows the viral titer results of the transfection experiments described in the embodiments herein. HEK293 cells were transfected with plasmids containing E1A at 1:1, 2:1, or 3:1 molar ratios to each of the standard triple transfection plasmids ("3xTnfx"), indicated as 1xE1A, 2xE1A, or 3xE1A, respectively. Crude viral titers were analyzed by droplet digital PCR (ddPCR). [Figure 5A] Described in embodiments herein are novel and enhanced triple transfection plasmids for AAV production in E1-complementing producer cells: pLHI_Helper plasmid containing E1A, E2A, E4, and VA RNAs, pRep-Cap plasmid containing Rep and Cap, and pAAV-GOI plasmid containing inverted terminal repeat (ITR) sequences and a gene of interest (GOI). [Figure 5B] Figure 1 shows the viral titer results of the transfection experiments described in the embodiments herein. HEK293 cells were transfected with the standard triple transfection plasmid containing pHelper (shown in Figure 1) or the new and enhanced triple transfection plasmid containing pLHI_Helper for AAV2 and AAV9 production (shown in Figure 5). Crude viral titers were analyzed by droplet digital PCR (ddPCR). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0012] Although use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0013] As used herein, the terms "comprising" (and any variation or form of includes, such as "comprise" and "comprises"), "having" (and any variation or form of includes, such as "have" and "has"), "including" (and any variation or form of includes, such as "includes" and "include"), or "containing" (and any variation or form of includes, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0014] The use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular term recited is representative of examples and embodiments of the present disclosure that are not intended to be limited to the particular example referenced or cited, unless expressly stated otherwise.

[0015] As used herein, "between" is a range that includes the ends of the range. For example, a number between x and y explicitly includes the numbers x and y, as well as any number that falls within the range between x and y.

[0016] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value. Typically, the term is meant to encompass a variation of less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0017] Adeno-associated virus (AAV) has been used as the vector of choice for gene therapy in over 120 clinical trials worldwide. To meet the rapidly growing demand for recombinant AAV, improvements to the current standard for AAV production in E1-complementing producer cells, such as HEK293, PER.C6, or CAP® cells, are necessary. As described herein, the standard AAV production method involves transfection of three plasmids into E1-complementing producer cells: pHelper, containing E2A, E4, and VA; pRepCap, containing Rep and Cap; and pAAV, containing the gene of interest (GOI), referred to as "standard triple transfection" and shown in FIG. 1. E1-complementing producer cells naturally express the remaining helper genes E1A and E1B, and thus E1A and E1B are not included in pHelper.

[0018] Thus, it has been surprisingly discovered that transfection of E1A into E1-complementing producer cells, such as HEK293 cells, thus providing the additional E1A as found in nature, significantly improves AAV titer. The increase in titer was generally dose-dependent on the amount of E1A introduced into the E1-complementing producer cells. Thus, methods are provided herein for AAV production in E1-complementing producer cells with higher yields compared to the current standard triple transfection method.

[0019] In embodiments, the disclosure provides methods of producing adeno-associated virus (AAV) in E1-complementing producer cells, comprising: (a) transfecting an E1-complementing producer cell with one or more vectors comprising: (i) an E1A adenoviral helper gene; (ii) an adenoviral helper gene selected from E2A, E4, or both; (iii) a virus-associated, non-coding RNA (VA RNA); and (iv) an AAV gene selected from Rep, Cap, or both; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV.

[0020] As used herein, a "vector" or "expression vector" refers to a nucleic acid replicon, such as a plasmid, phage, virus, or cosmid, to which another nucleic acid segment may be attached to effect replication and / or expression of the attached nucleic acid segment in a host cell. The term "vector" includes episomal (e.g., plasmid) and non-episomal vectors. The term vector may also include synthetic vectors. Non-limiting examples of vectors include baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, etc.), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for a particular host of interest (e.g., mammalian cells such as E1-complementing producer cells described herein, including but not limited to HEK293 cells, PER.C6 cells, CAP® cells, and derivatives thereof). The vector may be introduced into a desired host cell, such as HEK293 cells, PER.C6 cells, CAP® cells, or derivatives thereof, by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection. The vector may include various regulatory elements, including, for example, constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like.

[0021] As used herein, "transfection" refers to the introduction of an exogenous nucleic acid molecule, e.g., a vector, into a cell. A "transfected" cell contains an exogenous nucleic acid molecule within the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule within the cell induces a phenotypic change within the cell. The transfected nucleic acid molecule may be integrated into the genomic DNA of the host cell and / or may be maintained by the cell either transiently or extrachromosomally long term. A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant," "transformed," or "transgenic" organism. Numerous transfection techniques are generally known in the art and include a variety of chemical and physical methods, such as electroporation, cell injection, calcium phosphate exposure, liposome or polymer-based carrier systems, and the like. See, e.g., Graham et al., Virology 52:456 (1973), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989), Davis et al., Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al., Gene 13:197 (1981). Using such techniques, one or more exogenous nucleic acid molecules, such as one or more vectors for producing the AAV described herein, can be introduced into a suitable host cell, such as a HEK293 cell.

[0022] "Nucleic acid", "nucleic acid molecule", "polynucleotide", or "oligonucleotide" refers to a polymeric compound containing covalently linked nucleotides. The term "nucleic acid" includes RNA and DNA, both of which can be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, or miRNA.

[0023] In embodiments, a nucleic acid herein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to a reference nucleic acid (or a fragment of a reference nucleic acid). In embodiments, a polypeptide herein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to a reference polypeptide (or a fragment of a reference polypeptide). For example, when referring to the E1A gene, one skilled in the art will understand that E1A encompasses nucleic acids having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with the wild-type adenovirus E1A gene, provided that the function of the E1A gene with respect to AAV production is not affected. As used herein, the term "sequence identity" or "percent identity" in the context of a nucleic acid refers to the percentage of nucleotides that are the same when nucleic acid sequences are aligned over a specified comparison window. Methods of alignment for determining sequence identity are well known and can be performed using publicly available databases such as BLAST.

[0024] "Gene" refers to a nucleic acid that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. In some embodiments, "gene" also refers to non-coding nucleic acid fragments that can function as regulatory sequences preceding (i.e., 5') and following (i.e., 3') the coding sequence.

[0025] As used herein, the term "adeno-associated virus (AAV)" refers to a small-sized, replication-deficient, non-enveloped virus that contains single-stranded DNA of the Parvoviridae and Dependoparvovirus genera. More than 10 adeno-associated virus serotypes have been identified to date, with serotype AAV2 being the best characterized. Other non-limiting examples of AAV serotypes are ANC80, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In addition to these serotypes, AAV pseudotypes have been developed. AAV pseudotypes contain a capsid of a first serotype and a genome of a second serotype (e.g., pseudotype AAV2 / 5 corresponds to an AAV with a genome of serotype AAV2 and a capsid of AAV5).

[0026] As used herein, the term "adenovirus" refers to a non-enveloped virus with an icosahedral nucleocapsid containing double-stranded DNA of the Adenoviridae family. More than 50 adenovirus subtypes have been isolated from humans, and many additional subtypes have been isolated from other mammals and birds. These subtypes belong to the Adenoviridae family, which is currently divided into two genera: Mastadenovirus and Aviadenovirus. All adenoviruses are morphologically and structurally similar. However, in humans, adenoviruses show different immunological properties and are therefore classified into serotypes. Two human serotypes of adenovirus, Ad2 and Ad5, have been extensively studied and provide most of the general information about adenoviruses.

[0027] As used herein, the term "E1 complementing producer cell" refers to a cell line, typically a human cell line, into whose genome the adenoviral E1A and E1B genes are integrated or inserted. E1 complementing producer cells can naturally express E1A and E1B and are therefore suitable for AAV production without the need for exogenous E1A and / or E1B genes. The E1A and E1B genes may be suitably integrated into the E1 complementing producer cell as part of a longer sequence, e.g., containing additional adenoviral sequences, or the E1A and E1B genes may be suitably integrated into the E1 complementing producer cell without any additional adenoviral sequences, i.e., only the minimal necessary coding sequences for expressing E1A and E1B are integrated. The integration of adenoviral genes into mammalian cells, e.g., human cells, is known in the art. E1 complementing producer cells may be derived from any suitable cell line. Exemplary E1 complementation producer cells include, but are not limited to, HEK293 cells, 911 cells, pTG6559 cells, PER.C6 cells, GH329 cells, N52.E6 cells (also known as "CAP®" cells), HeLa-E1 cells, UR cells, VLI-293 cells, Ac51 cells, Ac139 cells, and variants and derivatives thereof. E1 complementation producer cells are further described, for example, in Kovesdi et al., Viruses 2(8):1681-1703 (2010) and Farson et al., Mol Ther 14(2):305-311 (2006). In some embodiments, the E1 complementation producer cells of the present disclosure are HEK293 cells, PER.C6 cells, or CAP® cells.

[0028] As used herein, the term "human embryonic kidney 293 (HEK293) cells" refers to a cell line originally derived from human embryonic kidney cells and containing approximately 4.5 kb of the Ad5 genome, including the E1A and E1B genes. HEK293 cells are exemplary E1 complementation producer cells described herein. Variants of HEK293 cells have been developed, including, for example, HEK293S, HEK293T, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEK293MSR, and HEK293A, each of which has different characteristics and all of which contain the E1A and E1B genes. As used herein, the term "HEK293 cells" encompasses all HEK293 cell variants and derivatives, including but not limited to the variants described herein. For example, the HEK293T cell line expresses a temperature-sensitive allele of the SV40 T antigen, which allows the amplification of vectors containing the SV40 origin. See, e.g., Yuan et al., "The Scattered Twelve Tribes of HEK293," Biomed Pharmacol J 2018;11(2).

[0029] As used herein, the term "PER.C6 cells" refers to a cell line derived from human fetal retina cells transformed with the E1 region of Ad5. PER.C6 cells naturally express E1A and E1B and are exemplary E1 complementation producer cells described herein. As used herein, the term "PER.C6 cells" encompasses all PER.C6 cell variants and derivatives. PER.C6 cells are further described, for example, in Fallaux et al., Human Gene Ther 9(13):1909-1917 (1998).

[0030] As used herein, the term "CAP® cells" (also referred to herein as "CEVEC amnion cell producer cells" or "N52.E6 cells") refers to a cell line derived from primary human amnion cells transformed with the E1 region of Ad5 (accessible under RRID: CVCL_IQ88). CAP® cells naturally express E1A and E1B and are exemplary E1 complementation producer cells described herein. CAP® cell variants include, but are not limited to, CAP-T (Accession Number: CVCL_WI61), which expresses the large T antigen of SV40. See, e.g., Wolfel et al., BMC Proceedings 5(Suppl 8):P133 (2011). As used herein, the term "CAP® cells" encompasses all CAP® cell variants and derivatives.

[0031] As used herein, the term "E1A" refers to the adenovirus early region 1A (E1A) gene, which is expressed during adenovirus replication at the early stage of viral life. The E1A gene produces multiple protein isoforms, including, but not limited to, 289R, 243R, 217R, 171R, and 55R, with 289R, 243R, and 171R being the major isoforms of the Ad5 E1A gene. As used herein, the term "E1B" refers to the adenovirus early region 1B (E1B) gene, which expresses two proteins: a 55 kDa protein and a 19 kDa protein, referred to as "E1B-19k" and "E1B-55k," respectively. As described herein, E1A, E1B-19k, and E1B-55k are adenovirus helper genes expressed by E1 complementing producer cells. Because E1A, E1B-19k, and E1B-55k are produced by E1-complementing producer cells in sufficient amounts for AAV production, traditionally, the E1A and E1B genes are not exogenously introduced into E1-complementing producer cells when producing AAV.

[0032] As used herein, the term "E2A" refers to the adenovirus E2A helper gene, which encodes a 72 kDa DNA-binding protein that regulates viral replication. As used herein, the term "E4" refers to the adenovirus E4 helper gene, which encodes proteins that regulate viral replication and protein expression, including E4 ORF3 and E4 ORF6. In embodiments, E4 includes one or both of E4orf3 and E4orf6. As used herein, the term "virus-associated, non-coding RNA (VA RNA)" refers to a gene that plays a role in regulating translation and is involved in the VAI (or VA I or VA RNAI) and VAII (or VA II The term "VA" refers to adenoviral non-coding RNA, including adenoviral non-coding RNA (VA RNA), including adenoviral non-coding RNA (E2A, E4, or VA RNA II). In embodiments, VA RNA includes one or both of VAI and VAII. In the context of AAV production, the adenoviral genes E2A, E4, and VA RNA mediate AAV replication. As described herein, the current method for producing AAV in E1-complementing producer cells involves transfecting the E1-complementing producer cells with a pHelper plasmid that includes the E2A, E4, and VA RNA genes.

[0033] As used herein, the term "Rep" refers to an AAV coding region that encodes viral replication proteins, or functional homologs thereof, that are collectively required to replicate the viral genome. Functional homologs of AAV Rep include, for example, the Rep coding region from human herpesvirus 6 (HHV-6), which is also known to mediate AAV DNA replication. The Rep coding region encodes at least the AAV Rep78, Rep68, Rep52, and Rep40-encoding genes, or functional homologs thereof. The Rep coding region can be derived from any AAV serotype, for example, as described herein. The Rep coding region need not contain all wild-type genes, but may be modified (e.g., by insertion, deletion, or mutation of one or more nucleotides), so long as the Rep genes present provide sufficient replication functions when expressed in an E1-complementing producer cell.

[0034] As used herein, the term "Cap" refers to the AAV coding region that encodes the viral capsid protein or a functional homolog thereof. Non-limiting examples of capsid proteins are the AAV capsid proteins VP1, VP2, and VP3. The Cap genes described herein can be from any AAV serotype or combination of AAV serotypes.

[0035] In some embodiments, the E1 complementation producer cell is transfected with one or more vectors comprising (i) E1A, (ii) one or both of E2A and E4, (iii) VA RNA, and (iv) one or more of Rep and Cap. In certain embodiments, (i), (ii), (iii), and (iv) are on a single vector. In further embodiments, (i), (ii), (iii), and (iv) are on two or more vectors. Suitably, (i), (ii), and (iii) may be on a first vector and (iv) may be on a second vector. In embodiments, (ii) comprises both E2A and E4. In further embodiments, (iv) comprises both Rep and Cap. For example, a first vector may comprise E1A, E2A, E4, and VA RNA, and a second vector may comprise Rep and Cap. Suitably, (i) may be on a first vector, (ii) and (iii) may be on a second vector, and (iv) may be on a third vector. For example, the first vector may include E1A, the second vector may include E2A, E4, and VA RNA, and the third vector may include Rep and Cap. Suitably, any one of (i), (ii), (iii), and (iv) may be on a separate vector, for example, each of (i), (ii), (iii), and (iv) may be on a separate vector. For example, the first vector may include E1A, the second vector may include E2A and E4, the third vector may include VA RNA, and the fourth vector may include Rep and Cap. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0036] In some embodiments, E1A is transfected into E1-complementing producer cells in an amount greater than any one of E2A, E4, VA RNA, Rep, and Cap. As discussed herein, it has been unexpectedly discovered that AAV titers are dose-dependent on the amount of E1A expressed in E1-complementing producer cells. Thus, in embodiments, E1A is introduced into E1-complementing producer cells in an amount that is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater than 10-fold greater than any one of E2A, E4, VA RNA, Rep, and Cap. Methods of increasing expression of a particular gene may include, for example, increasing the amount of the gene introduced into the cell and increasing the transcription level of the gene in the cell. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0037] Suitably, the E1A-containing vector may be transfected into an E1 complementation producer cell at a ratio of about 1:1 to about 10:1 to each of the E2A, E4, VA RNA, Rep, and / or Cap-containing vectors, or about 1:1 to about 9:1 to each of the E2A, E4, VA RNA, Rep, and / or Cap-containing vectors, or about 1:1 to about 8:1, or about 1:1 to about 7:1, or about 1:1 to about 6:1, or about 1:1 to about 5:1, or about 1:1 to about 4:1, or about 1:1 to about 3:1, or about 1:1 to about 2:1, or about 1:1 to about 1.5:1 to each of the E2A, E4, VA RNA, Rep, and / or Cap-containing vectors. In some embodiments, the ratio of E1A-containing vector to each of the E2A, E4, VA RNA, Rep, and / or Cap-containing vectors transfected into an E1 complementation producer cell is 1:1. In a further embodiment, the ratio of E1A-containing vector to each of E2A, E4, VA RNA, Rep, and / or Cap-containing vectors transfected into the E1 complementing producer cell is 2: 1. In yet a further embodiment, the ratio of E1A-containing vector to each of E2A, E4, VA RNA, Rep, and / or Cap-containing vectors transfected into the E1 complementing producer cell is 3: 1. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0038] In some embodiments, the expression level of E1A in E1 complementation producer cells (e.g., HEK293 cells, PER.C6 cells, or CAP® cells) is higher compared to each of E2A, E4, VA RNA, Rep, and / or Cap. Suitably, each of E1A, E2A, E4, VA RNA, Rep, and / or Cap can be operably linked to one or more promoters. In some embodiments, each of E1A, E2A, E4, VA RNA, Rep, and / or Cap is operably linked to a separate promoter. As used herein, the terms "operably linked," "in operable combination," and "in operable order" mean that a polynucleotide of interest (e.g., E1A) is linked to a regulatory element (e.g., a promoter) in a manner that allows expression of the polynucleotide of interest. As used herein, the terms "promoter," "promoter sequence," and "promoter region" refer to a polynucleotide that can bind RNA polymerase and initiate transcription of a downstream coding or non-coding gene sequence. Promoters include constitutive promoters, which allow continuous transcription of their associated genes, and inducible promoters, which can be switched from an off state to an on state upon addition of an inducer molecule. Promoters can be classified as "strong" or "weak" based on their affinity for RNA polymerase and / or sigma factors. Strong promoters have a high transcription initiation rate compared to weak promoters.Non-limiting examples of promoters include the spleen-limited focus-forming virus (SFFV) promoter, human polypeptide chain elongation factor (EF1α) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin C (UBC) promoter, cytomegalovirus (CMV) promoter, chicken beta-actin (CBA) promoter, tetracycline-controlled transactivator system (also known as the "Tet-On" system or tTA-dependent system), or reverse tetracycline-controlled transactivator system (also known as the "Tet-Off" system or rtTA-dependent system), Rous sarcoma virus extended repeat (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, actin promoter, cytokeratin 14 promoter, or cytokeratin 18 promoter. In some embodiments, the promoter operably linked to E1A is a stronger promoter than the promoter operably linked to any of E2A, E4, VA RNA, Rep, and Cap.

[0039] Suitably, a promoter operably linked to E1A can provide 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold higher expression than a promoter operably linked to any of E2A, E4, VA RNA, Rep, and Cap. In further embodiments, a promoter operably linked to E1A provides substantially the same expression level as a promoter operably linked to any of E2A, E4, VA RNA, Rep, and Cap. As used herein, "substantially similar" or "substantially the same" expression level means that the expression level is within 20%, within 15%, within 10%, within 5%, or within 1% of a reference expression level.

[0040] In some embodiments, E1A is operably linked to a promoter and further operably linked to an enhancer, and each of E2A, E4, VA RNA, Rep, and / or Cap is preferably not bound to an enhancer. As used herein, the term "enhancer" refers to a regulatory element that activates transcription to a higher level than in their absence, for example by recruiting a transcription factor. Enhancers can activate promoter transcription over long distances (e.g., up to 1 Mbp away from the transcription start site) and independent of orientation. Non-limiting examples of enhancers include CMV enhancer, alpha-fetoprotein MERII enhancer, MCK enhancer, or SV40 polyA enhancer. Preferably, E1A operably linked to a promoter and enhancer can provide a higher expression level compared to E2A, E4, VA RNA, Rep, and / or Cap not bound to an enhancer.

[0041] Suitably, one or more of the vectors may contain an origin of replication to increase the number of plasmids produced by the host cell, which results in increased levels of proteins encoded by genes on one or more of the vectors. Exemplary origins of replication include, but are not limited to, the SV40 origin of replication, the Epstein-Barr (EBV) origin of replication, and others known to those skilled in the art.

[0042] In some embodiments, the number of copies of E1A, referred to herein as "copy number," present on one or more vectors is higher than the number of copies of each of E2A, E4, VA RNA, Rep, and / or Cap on one or more vectors. In one example, a single vector can contain two or more copies of E1A and a single copy of each of E2A, E4, VA RNA, Rep, and Cap. In another example, for each copy of E2A, E4, VA RNA, Rep, and Cap present on one or more vectors, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of E1A are present on the one or more vectors. Suitably, the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and / or Cap on one or more vectors can be about 1:1 to about 10:1, or about 1:1 to about 9:1, or about 1:1 to about 8:1, or about 1:1 to about 7:1, or about 1:1 to about 6:1, or about 1:1 to about 5:1, or about 1:1 to about 4:1, or about 1:1 to about 3:1, or about 1:1 to about 2:1, or about 1:1 to about 1.5:1. In some embodiments, the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and / or Cap on one or more vectors is about 1:1. In further embodiments, the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and / or Cap on one or more vectors is about 2:1. In still further embodiments, the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and / or Cap on the one or more vectors is about 3:1.

[0043] In some embodiments, the method comprises transfecting additional helper genes into the E1 complementing producer cell. Thus, in embodiments, the one or more vectors further comprise helper genes selected from (i) E1A, (ii) one or both of E2A and E4, (iii) VA RNA, (iv) one or both of Rep and Cap, and (v) E1B, NP1, NS2, or combinations thereof. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0044] As discussed herein, E1B is an adenovirus helper gene that encodes E1B-19k and E1B-55k helper proteins for AAV production. As used herein, the terms "NP1" and "NS2" refer to human bocavirus (HBoV1) genes that encode NP1 and NS2 nonstructural proteins, respectively. NP1 and NS2 have been discovered as helper genes for AAV replication and have been shown to enhance AAV titers when co-expressed with conventional adenovirus helper genes for E1 complementing producer cells, i.e., E2A, E4, and VA RNA as described herein. See, e.g., Wang et al., Mol Ther Methods Clin Dev 11:40-51 (2018). In some embodiments, transfecting one or more of E1B, NP1, and NS2 in combination with (i), (ii), (iii), and (iv) described herein further increases AAV titer in the E1 complementing producer cell. In certain embodiments, (v) comprises EB1. In further embodiments, (v) comprises NP1 and NS2. In yet further embodiments, (v) comprises E1B, NP1, and NS2. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0045] In some embodiments, (i), (ii), (iii), (iv), and (v) are on a single vector. In further embodiments, (i), (ii), (iii), (iv), and (v) are on two or more vectors. For example, (i), (ii), and (iii) can be on a first vector, (iv) can be on a second vector, and (v) can be on a third vector. In another example, (i), (ii), (iii), and (v) can be on a first vector, and (iv) can be on a second vector. In a further example, (i), (ii), and (iii) can be on a first vector, and (iv) and (v) can be on one or more additional vectors. In yet a further example, (i) and (v) can be on a first vector, and (ii), (iii), and (iv) can be on one or more additional vectors. Suitably, any one of (i), (ii), (iii), (iv), and (v) may be on a separate vector, for example, each of (i), (ii), (iii), (iv), and (v) may be on a separate vector. In an embodiment, a first vector comprises E1A, E2A, E4, and VA RNA, and a second vector comprises Rep and Cap. In an additional embodiment, a first vector comprises E1A, E2A, E4, and VA RNA, a second vector comprises Rep and Cap, and a third vector comprises E1B, NP1, NS2, or a combination thereof.

[0046] E1 complementation producer cells (e.g., HEK293 cells, PER.C6 cells, or CAP® cells) can be cultured using any suitable device, facility, and method described herein after transfection with helper genes selected from (i) E1A, (ii) one or both of E2A and E4, (iii) VA RNA, (iv) one or both of Rep and Cap, (v) E1B, NP1, NS2, or a combination thereof, and / or a GOI. Suitably, expression of (i), (ii), (iii), (iv), and (v) can be induced during culture. In certain embodiments, the promoter of any of (i), (ii), (iii), (iv), and / or (v) is a constitutive promoter. In further embodiments, the promoter of any of (i), (ii), (iii), (iv), and / or (v) is an inducible promoter and requires an inducer molecule to activate transcription as described herein. In one embodiment, the expression level of a gene under the control of an inducible promoter can be regulated by adjusting the amount of an inducer molecule. In another embodiment, the same inducible promoter can be regulated using two or more inducer molecules to result in different expression levels. In a further embodiment, the promoter operably linked to (i) is a stronger promoter than the promoter operably linked to (ii), (iii), (iv), and / or (v). For example, (i) can be under the control of a relatively strong promoter, such as a CMV or SV40 promoter, and (ii), (iii), (iv), and / or (v) can be under the control of a relatively weak promoter, such as a UBC or PGK promoter. In a further embodiment, the promoter operably linked to (i) is induced by a different inducer molecule than the promoter operably linked to (ii), (iii), (iv), and / or (v).For example, (i) may be under the control of a Tet-On system that is inducible by analogs such as tetracycline (Tet) or doxycycline (Dox), and (ii), (iii), (iv), and / or (v) may be under the control of a constitutive promoter such as CMV, SV40, UBC, or PGK, and the level of E1A expression can be regulated by the amount of inducer molecule (e.g., Tet or Dox) added to the cell culture. Thus, the amount of inducer molecule added to the cell culture can determine the relative amounts of (i), (ii), (iii), (iv), and / or (v) expressed in the cell culture.

[0047] In embodiments in which the one or more vectors include (i), (ii), (iii), and (iv), culturing includes regulating expression of (i), (ii), (iii), and (iv), such that E1A is expressed in a ratio of 1:1 to about 10:1 for each of (ii), (iii), and (iv), or from about 1:1 to about 9:1 for each of (ii), (iii), and (iv), or from about 1:1 to about 8:1, or from about 1:1 to about 7:1, or from about 1:1 to about 6:1, or from about 1:1 to about 5:1, or from about 1:1 to about 4:1, or from about 1:1 to about 3:1, or from about 1:1 to about 2:1, or from about 1:1 to about 1.5:1. Suitably, the ratio of expression of E1A to expression of each of (ii), (iii), and (iv) is about 1:1. In a further embodiment, the ratio of expression of E1A to each of (ii), (iii), and (iv) is about 2: 1. In yet a further embodiment, the ratio of expression of E1A to each of (ii), (iii), and (iv) is about 3:1.

[0048] In embodiments in which the one or more vectors include (i), (ii), (iii), (iv), and (v), the culturing includes regulating expression of (i), (ii), (iii), (iv), and (v), such that E1A is expressed in a ratio of from 1:1 to about 10:1, or from about 1:1 to about 9:1, or from about 1:1 to about 8:1, or from about 1:1 to about 7:1, or from about 1:1 to about 6:1, or from about 1:1 to about 5:1, or from about 1:1 to about 4:1, or from about 1:1 to about 3:1, or from about 1:1 to about 2:1, or from about 1:1 to about 1.5:1 to each of (ii), (iii), (iv), and (v). Suitably, the ratio of expression of E1A to each of (ii), (iii), (iv) and (v) is about 1:1, or about 2:1, or about 3:1.

[0049] In some embodiments, one or more of the vectors further comprises a gene of interest (GOI). Suitably, the GOI is on a separate vector from each of (i), (ii), (iii), (iv), and (v). Thus, in embodiments, the method further comprises transfecting the E1 complementation producer cell with a vector comprising the GOI, wherein the GOI is on a separate vector from each of (i), (ii), (iii), (iv), and (v). Suitably, the vector comprising (i) is transfected into the E1 complementation producer cell at a ratio of about 1:1 to about 10:1 relative to the vector comprising the GOI, or about 1:1 to about 9:1 relative to the vector comprising the GOI, or about 1:1 to about 8:1, or about 1:1 to about 7:1, or about 1:1 to about 6:1, or about 1:1 to about 5:1, or about 1:1 to about 4:1, or about 1:1 to about 3:1, or about 1:1 to about 2:1, or about 1:1 to about 1.5:1. Suitably, the ratio of vector comprising (i) to vector comprising the GOI transfected into the E1 complementation producer cell is about 1:1, about 2:1, or about 3:1. Suitably, the E1 complementation producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0050] The vector containing the GOI may contain two inverted terminal repeat (ITR) sequences flanking the GOI. As known in the art, these ITR sequences are single-stranded nucleotide sequences followed downstream by their reverse complement. The ITR sequences represent the minimal sequences required for replication, rescue, packaging, and integration of the AAV genome.

[0051] Generally, as used herein, the term "GOI" refers to a heterologous gene, i.e., a gene that is not normally linked together and / or is not normally associated with a particular host cell. In some embodiments, the heterologous gene is a construct whose coding sequence is not itself found in nature (e.g., a synthetic sequence with codons different from the natural gene). Suitably, the GOI may be a reporter gene, a selection gene, or a gene of therapeutic interest.

[0052] As referred to herein, a "reporter gene" is a gene that, upon expression, confers a phenotype on a cell that can be easily identified and measured. In some embodiments, the reporter gene encodes a fluorescent protein. In further embodiments, the reporter gene comprises a selection gene.

[0053] As referred to herein, a "selection gene" is a gene that encodes a protein with enzymatic activity that confers to a host cell the ability to grow in a medium that otherwise lacks an essential nutrient. Alternatively or additionally, a selection gene may confer resistance to an antibiotic or drug to the host cell in which the selection gene is expressed. A selection gene may also be used to confer a particular phenotype to a host cell. If a host cell expresses the selection gene in order to grow in a selective medium, the gene is said to be a positive selection gene. Selection genes can also be used to select against host cells that contain a particular gene, and selection genes used in this manner are referred to as negative selection genes.

[0054] As used herein, the term "therapeutic gene" refers to any functionally related nucleotide sequence. Thus, the therapeutic gene of the present disclosure can include any desired gene that encodes a protein that is missing or missing from the target cell genome, or encodes a non-native protein with a desired biological or therapeutic effect (e.g., antiviral function), or the sequence can correspond to a molecule with antisense or ribozyme function. Representative (non-limiting) examples of suitable therapeutic genes include those used to treat inflammatory, autoimmune, chronic, and infectious diseases, including disorders such as AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, and the like; various blood disorders, including various anemias, thalassemias, and hemophilias; genetic defects such as cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, and the like. Several antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of mRNA) useful for antisense treatment of cancer and viral diseases have been described in the art and are also examples of suitable therapeutic genes.

[0055] In additional embodiments, the disclosure provides a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A operably linked to a first promoter, (ii) a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter, and (iii) a third vector comprising Rep and Cap, both operably linked to a third promoter, wherein the transfection ratio of (i) to each of (ii) and (iii) is from about 1:1 to about 3:1; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV. Suitably, one or more of the first, second or third vector further comprises a helper gene selected from E1B, NP1, NS2, or a combination thereof. Suitably, E1B is E1B-19k or E1B-55k. Suitably, the helper gene comprises a combination of NP1 and NS2. In an embodiment, the method further comprises transfecting an E1 complementing producer cell with a vector comprising the GOI. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0056] In a further embodiment, the disclosure provides a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A operably linked to a first promoter and enhancer, (ii) a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter, and (iii) a third vector comprising Rep and Cap, each operably linked to a third promoter; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV, wherein E1A is expressed in a ratio of about 1:1 to about 3:1 relative to each of E2A, E4, VA RNA, Rep, and Cap; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV. Suitably, one or more of the first, second or third vector further comprises a helper gene selected from E1B, NP1, NS2, or a combination thereof. Suitably, E1B is E1B-19k or E1B-55k. Suitably, the helper gene comprises a combination of NP1 and NS2. In an embodiment, the method further comprises transfecting an E1 complementing producer cell with a vector comprising the GOI. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0057] In still further embodiments, the disclosure provides a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A, E2A, E4, and VA RNA, all operably linked to a first promoter, and (ii) a second vector comprising Rep and Cap, operably linked to a second promoter, wherein the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and Cap is from about 1:1 to about 3:1; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV. Suitably, one or both of the first or second vectors further comprise a helper gene selected from E1B, NP1, NS2, or a combination thereof. Suitably, E1B is E1B-19k or E1B-55k. Suitably, the helper genes comprise a combination of NP1 and NS2. In an embodiment, the method further comprises transfecting an E1 complementing producer cell with a vector comprising the GOI. Suitably, the E1 complementing producer cell is a HEK293 cell, a PER.C6 cell, or a CAP® cell.

[0058] In embodiments, provided herein is a method of treatment in a subject in need of treatment with AAV, comprising transfecting an E1 complementation producer cell (HEK293 cell, PER.C6 cell, or CAP® cell) with one or more vectors comprising (i) an E1A adenoviral helper gene, (ii) an adenoviral helper gene selected from E2A, E4, or both, (iii) a virus-associated, non-coding RNA (VA RNA), and (iv) an AAV gene selected from Rep, Cap, or both, producing AAV, harvesting the AAV, and administering the AAV to the subject. Methods of producing AAV are further described herein. Suitably, the method is used to treat a subject having a GOI, e.g., a gene of therapeutic interest. Administration to a human subject can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.

[0059] As discussed herein, the methods of the disclosure advantageously provide higher titers of AAV produced by E1-complementing producer cells compared to methods that do not include transfecting E1-complementing producer cells with E1A. In embodiments, the methods of the disclosure provide at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more titers of AAV compared to methods that do not include transfecting E1-complementing producer cells with E1A. Suitably, the E1-complementing producer cells are HEK293 cells, PER.C6 cells, or CAP® cells.

[0060] The method for producing AAV can be used in a continuous manufacturing system. Culture conditions for E1 complementation producer cells, such as HEK293 cells, PER.C6 cells, or CAP® cells, are known in the art. Typically, HEK293 cells, PER.C6 cells, and CAP® cells are suspension cultures, which are grown, for example, in culture flasks or large suspension tanks that allow for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize stirring or agitation mechanisms to provide adequate mixing. Media and conditions for maintaining cells in suspension are known to those of skill in the art. In an exemplary embodiment, the use of suspension cell cultures allows for large-scale production of AAV with high productivity and long-term culture conditions, allowing multiple recovery of AAV for each batch of starting cells.

[0061] The production method may utilize any suitable reactor, including, but not limited to, stirred tank, air-lift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, a "reactor" may include a fermenter or fermentation unit, or any other reaction vessel, and the term "reactor" is used synonymously with "fermenter." For example, in some embodiments, an exemplary bioreactor unit may perform one or more, or all of the following: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, may include multiple reactors within the unit, e.g., the unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit and / or facility, and / or may include multiple units with single or multiple reactors within a facility. In various embodiments, the bioreactors may be suitable for batch, semi-fed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactors may have a volume of about 100 mL to about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, and the like. Examples of reactors include reactors having volumes of 100 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0062] In embodiments, and unless otherwise stated herein, the devices, facilities, and methods described herein may also include any suitable unit operations and / or equipment not specifically mentioned, such as operations and / or equipment for the separation, purification, and isolation of such products. Any suitable facilities and environments may be used, such as conventional stick-built facilities, modular equipment, mobile and temporary facilities, or any other suitable construction, facility, and / or layout. For example, in some embodiments, modular clean rooms may be used. Furthermore, unless otherwise stated, the devices, systems, and methods described herein may be housed and / or performed in a single location or facility, or alternatively, may be housed and / or performed in separate or multiple locations and / or facilities.

[0063] All references cited herein, including patents, patent applications, articles, textbooks, and the like, as well as the references cited therein, to the extent they have not already been cited, are hereby incorporated by reference in their entirety.

[0064] Additional Exemplary Embodiments Embodiment 1 is a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with one or more vectors comprising: (i) an E1A adenoviral helper gene; (ii) an adenoviral helper gene selected from E2A, E4, or both; (iii) a virus-associated, non-coding RNA (VA RNA); and (iv) an AAV gene selected from Rep, Cap, or both; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV.

[0065] Embodiment 2 includes the method of embodiment 1, wherein (i), (ii), (iii), and (iv) are on a single vector.

[0066] Embodiment 3 includes the method of embodiment 1, wherein (i), (ii), (iii), and (iv) are on two or more vectors.

[0067] Embodiment 4 includes the method of embodiment 3, wherein (i), (ii), and (iii) are on a first vector and (iv) is a second vector.

[0068] Embodiment 5 includes the method of embodiment 3, wherein (i) is on a first vector, (ii) and (iii) are on a second vector, and (iv) is on a third vector.

[0069] Embodiment 6 includes the method of embodiment 3, wherein each of (i), (ii), (iii), and (iv) is on a separate vector.

[0070] Embodiment 7 includes the method of embodiment 5 or 6, wherein the vector containing (i) is transfected into the E1 complementation producer cell in a ratio of about 1:1 to about 5:1 to each of the vectors containing (ii), (iii), and (iv).

[0071] Embodiment 8 includes the method of embodiment 7, wherein the ratio of vector containing (i) to each of the vectors containing (ii), (iii), and (iv) transfected into the E1 complementation producer cell is 1:1.

[0072] Embodiment 9 includes the method of embodiment 7, wherein the ratio of vector containing (i) to each of the vectors containing (ii), (iii), and (iv) transfected into the E1 complementation producer cell is 2:1.

[0073] Embodiment 10 includes the method of embodiment 7, wherein the ratio of vector containing (i) to each of the vectors containing (ii), (iii), and (iv) transfected into the E1 complementation producer cell is 3:1.

[0074] Embodiment 11 includes the method of any one of Embodiments 1-10, wherein (i), (ii), (iii), and (iv) are operably linked to one or more promoters.

[0075] Embodiment 12 includes the method of embodiment 11, wherein each of (i), (ii), (iii), and (iv) is operably linked to a separate promoter.

[0076] Embodiment 13 includes the method of embodiment 12, wherein the promoter operably linked to (i) provides substantially the same expression level as compared to promoters operably linked to (ii), (iii), and (iv).

[0077] Embodiment 14 includes the method of embodiment 12, wherein the promoter operably linked to (i) provides at least two-fold higher expression compared to promoters operably linked to (ii), (iii), and (iv).

[0078] Embodiment 15 includes the method of embodiment 12, wherein the promoter operably linked to (i) provides at least three-fold higher expression compared to promoters operably linked to (ii), (iii), and (iv).

[0079] Embodiment 16 includes the method of any one of embodiments 11-15, wherein (i) is operably linked to a promoter and further operably linked to an enhancer, and (ii), (iii), and (iv) are not operably linked to an enhancer.

[0080] Embodiment 17 includes the method of any one of embodiments 1 to 16, wherein the copy number ratio of (i) to each of (ii), (iii), and (iv) on the one or more vectors is from about 1:1 to about 5:1.

[0081] Embodiment 18 includes the method of embodiment 17, wherein the copy number ratio of (i) to each of (ii), (iii), and (iv) on the one or more vectors is 1:1.

[0082] Embodiment 19 includes the method of embodiment 17, wherein the copy number ratio of (i) to each of (ii), (iii), and (iv) on the one or more vectors is 2:1.

[0083] Embodiment 20 includes the method of embodiment 17, wherein the copy number ratio of (i) to each of (ii), (iii), and (iv) on the one or more vectors is 3:1.

[0084] Embodiment 21 includes the method of any one of embodiments 1-20, wherein (ii) includes E2A and E4.

[0085] Embodiment 22 includes the method of any one of embodiments 1-21, wherein (iv) includes Rep and Cap.

[0086] 23. The method of any one of embodiments 1-22, wherein the culturing comprises expressing E1A at a ratio of about 1:1 to about 5:1 to each of (ii), (iii), and (iv).

[0087] Embodiment 24 includes the method of embodiment 23, wherein the ratio of expression of E1A to each of (ii), (iii), and (iv) is 1:1, 2:1, or 3:1.

[0088] Embodiment 25 includes the method of any one of embodiments 1 to 24, wherein the one or more vectors comprise (v) a helper gene selected from E1B, NP1, NS2, or a combination thereof.

[0089] Embodiment 26 includes the method of embodiment 25, wherein E1B is E1B-19k or E1B-55k.

[0090] Embodiment 27 includes the method of embodiment 25, wherein (v) comprises NP1 and NS2.

[0091] Embodiment 28 includes the method of any one of embodiments 25 to 27, wherein (i) and (v) are on a single vector.

[0092] 28. The method of any one of embodiments 25-27, wherein (v) is on a separate vector from (i), (ii), (iii), and (iv).

[0093] Embodiment 30 includes the method of any one of claims 1 to 29, wherein one or more of the vectors further comprises a gene of interest.

[0094] Embodiment 31 includes the method of any one of embodiments 1-29, further comprising transfecting the E1 complementation producer cell with a vector containing the gene of interest, wherein the gene of interest is on a separate vector from (i), (ii), (iii), and (iv).

[0095] Embodiment 32 includes any one of the methods of embodiments 1-31, wherein the method produces at least 1.5-fold higher titers of AAV compared to a method that does not include transfecting an E1 complementing producer cell with an E1A adenoviral helper gene.

[0096] Embodiment 33 is a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell, comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A operably linked to a first promoter, (ii) a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter, and (iii) a third vector comprising Rep and Cap, both operably linked to a third promoter, wherein a transfection ratio of (i) to each of (ii) and (iii) is from about 1:1 to about 3:1; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV.

[0097] Embodiment 34 is a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A operably linked to a first promoter and enhancer, (ii) a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter, and (iii) a third vector comprising Rep and Cap, each operably linked to a third promoter; culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV, wherein E1A is expressed in a ratio of about 1:1 to about 3:1 relative to each of E2A, E4, VA RNA, Rep, and Cap; and purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV.

[0098] Embodiment 35 is a method of producing adeno-associated virus (AAV) in an E1-complementing producer cell, comprising: (a) transfecting an E1-complementing producer cell with (i) a first vector comprising E1A, E2A, E4, and VA RNA, all operably linked to a first promoter, and (ii) a second vector comprising Rep and Cap, operably linked to a second promoter, wherein the copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and Cap is from about 1:1 to about 3:1; (b) culturing the transfected E1-complementing producer cell under conditions suitable for producing AAV; and (c) purifying the AAV from the cultured E1-complementing producer cell, thereby obtaining AAV.

[0099] Embodiment 36 includes the method of embodiment 33 or 34, wherein one or more of the first, second, or third vectors further comprises a helper gene selected from E1B, NP1, NS2, or a combination thereof.

[0100] Embodiment 37 includes the method of embodiment 35, wherein one or both of the first or second vectors further comprises a helper gene selected from E1B, NP1, NS2, or a combination thereof.

[0101] Embodiment 38 includes the method of any one of embodiments 33-35, further comprising transfecting the E1 complementation producer cell with a vector comprising a helper gene selected from E1B, NP1, NS2, or a combination thereof.

[0102] Embodiment 39 includes the method of any one of embodiments 36-38, wherein E1B is E1B-19k or E1B-55k.

[0103] Embodiment 40 includes the method of any one of embodiments 36-38, wherein the helper genes include a combination of NP1 and NS2.

[0104] Embodiment 41 includes the method of any one of embodiments 33 to 40, further comprising transfecting the E1 complementation producer cell with a vector containing the gene of interest.

[0105] Embodiment 42 includes the method of any one of embodiments 1 to 41, wherein the E1 complementation producer cell is a HEK293 cell, a 911 cell, a pTG6559 cell, a PER.C6 cell, a GH329 cell, a N52.E6 (CAP®) cell, a HeLa-E1 cell, a UR cell, a VLI-293 cell, an Ac51 cell, an Ac139 cell, or a variant or derivative thereof.

[0106] Embodiment 43 includes the method of embodiment 42, wherein the E1 complementation producer cell is a HEK293 cell, a PER.C6 cell, or an N52.E6 (CAP®) cell.

[0107] Embodiment 44 includes the method of embodiment 43, wherein the E1 complementation producer cell is a HEK293 cell.

[0108]

[0046] Embodiment 45 includes the method of embodiment 44, wherein the HEK293 cells are HEK293S cells, HEK293T cells, HEK293F cells, HEK293FT cells, HEK293FTM cells, HEK293SG cells, HEK293SGGD cells, HEK293H cells, HEK293E cells, HEK293MSR cells, HEK293A cells, or combinations thereof.

[0109] Embodiment 46 includes the method of embodiment 45, wherein the HEK293 cells are HEK293T cells.

[0110] Embodiment 47 includes the method of embodiment 43, wherein the E1 complementation producer cells are PER.C6 cells.

[0111] Embodiment 48 includes the method of embodiment 43, wherein the E1 complementation producer cells are N52.E6 (CAP®) cells. EXAMPLES

[0112] Example 1. Screening of candidate helper genes for AAV production To identify new helper genes to improve AAV production by triple transfection, full-length cDNAs for E1A, E1B-19k, E1B-55k, and human bocavirus NP1 and NS2 were synthesized. cDNAs for additional helper genes were subcloned between the CMV promoter and bovine growth hormone polyadenylation (BGH poly(A)) signal in the pcDNA3.1 vector to allow overexpression. NP1 and NS2 were combined with an internal ribosome entry site (IRES) for dual expression in a single mRNA transcript. Candidate plasmids contained: E1A alone (SEQ ID NO:1), E1B-19k alone (SEQ ID NO:2), E1B-55k alone (SEQ ID NO:3), NP1-NS2 (SEQ ID NO:4), E1A+E1B-19k, E1A+19B-55k, E1B-19k+E1B-55k, or E1A+E1B-19k+E1B-55k.

[0113] To test these candidate plasmids, HEK293 cells were grown in 30 mL cultures in 125 mL shaker flasks and transfected with the standard triple transfection plasmid and the candidate helper plasmids shown in Figure 1. For the Rep / Cap plasmid, pRep2-Cap9 was used, and for the pAAV plasmid, pAAV-CMV-GFP was used for the production of AAV9.GFP virus. The same amount of empty vector pcDNA3.1 was co-transfected as a control. Each candidate helper plasmid was tested in duplicate.

[0114] Crude virus was harvested 3 days after transfection and subjected to droplet digital PCR (ddPCR) titer analysis to quantify viral genomes containing particles per milliliter (vg / mL). The results in Figure 2 showed that additional expression of E1A, or E1A+E1B isoforms significantly increased titers by nearly 100% compared to controls, while expression of E1B isoforms or bocavirus NP1 and NS2 had minimal improvement on AAV titers.

[0115] Example 2. Confirmation of E1A overexpression for AAV production The experiment in Example 1 was repeated under the following conditions: empty vector (control), E1A alone, E1B-19k alone, E1B-55k alone, NP1-NS2, and E1A+NP1-NS2, and the crude virus titer results were analyzed by ddPCR. As shown in Figure 3A, E1A alone or E1A+bocavirus NP1 and NS2 genes increased AAV titers by approximately 50%, while bocavirus genes alone had no effect.

[0116] To confirm the overexpression of E1A, Western blot analysis was performed to evaluate E1A protein expression (transfection conditions shown in the table in Figure 3B). As shown in Figure 3B, additional co-transfection of E1A expressed higher levels of all three isoforms of E1A protein (289R, 243R, and 171R) in samples 3, 4, 11, and 12, which had plasmid-transfected E1A, compared with samples 1 and 2, which contained only E1A protein endogenously expressed by HEK293 cells. β-actin levels were measured as a control, and no significant differences were found between the different conditions. Protein expression levels of Rep (Rep78 and Rep52 isoforms) and Cap (VP1, VP2, and VP3) were also determined, and no significant differences were detected between E1A-transfected samples and the control. Thus, E1A overexpression may improve AAV titers through mechanisms other than the regulation of Rep and Cap protein expression.

[0117] Example 3. Dose-dependent improvement of E1A for AAV production The E1A function for AAV titer improvement was further tested by titration of E1A plasmid at 1:1, 2:1, or 3:1 molar ratio to pHelper plasmid, shown as 1xE1A, 2xE1A, or 3xE1A, respectively, in the table in Figure 4A. Transfection and AAV production were performed in the same manner as in Example 1. As shown in Figure 4A, the increase in E1A overexpression was confirmed by Western blot analysis. As shown in Figure 4B, AAV9.GFP titer increased in an E1A dose-dependent manner.

[0118] Example 4. Novel and enhanced triple transfection for AAV production To simplify transfection experiments, the E1A expression cassette was subcloned from the pcDNA3.1-E1A plasmid of Example 1 into the standard pHelper vector shown in Figure 1. The new helper plasmid, designated "pLHI_Helper" (SEQ ID NO: 5), was transfected into HEK293 cells instead of conventional pHelper, as shown in Figure 5A. As shown in Figure 5B, compared to standard triple transfection, pLHI_Helper-mediated triple transfection significantly improved AAV2.GFP and AAV9.GFP production by 52.2% and 36.1%, respectively, over the control standard triple transfection using pHelper.

[0119] array Exemplary Plasmid Containing SEQ ID NO:1-E1A (pcDNA3.1-E1A)

[0120] SEQ ID NO:2 - An exemplary plasmid containing E1B-19k (pcDNA3.1-E1B-19K)

[0121] Exemplary Plasmid Containing SEQ ID NO:3-E1B-55k (pcDNA3.1-E1B-55K)

[0122] SEQ ID NO:4 - An exemplary plasmid containing NP1 and NS2 (pcDNA3.1-NP1-NS2)

[0123] SEQ ID NO:5 - An exemplary plasmid containing E1A, E2A, E4, and VA RNA (pLHI_Helper)

[0124] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on February 7, 2022, is named 0132-0156WO1_SL.txt and is 55,665 bytes in size.

Claims

1. 1. A method for producing adeno-associated virus (AAV) in an E1 complementation producer cell, comprising: a. the E1 complementation producer cell, i. E1A adenoviral helper gene, ii. an adenoviral helper gene selected from E2A, E4, or both; iii. Virus-associated, non-coding RNA (VA RNA); iv. AAV genes selected from Rep, Cap, or both; and b. culturing the transfected E1-complementing producer cells under conditions suitable for producing the AAV; c) purifying said AAV from said cultured E1-complementing producer cells, thereby obtaining said AAV.

2. 2. The method of claim 1, wherein (i), (ii), (iii), and (iv) are on a single vector; or (i), (ii), (iii), and (iv) are on two or more vectors.

3. (i), (ii), (iii), and (iv) are on two or more vectors; and (i), (ii), and (iii) are on a first vector and (iv) is on a second vector; (i) is on a first vector, (ii) and (iii) are on a second vector, and (iv) is on a third vector; or 2. The method of claim 1, wherein each of (i), (ii), (iii), and (iv) is on a separate vector.

4. The method of claim 3, wherein (i) is on a vector separate from (ii), (iii), and (iv), and the vector containing (i) is transfected into the E1 complementation producer cell in a ratio of 1:1 to 5:1 to each of the vectors containing (ii), (iii), and (iv), optionally the ratio is 1:1, 2:1, or 3:

1.

5. The method of any one of claims 1 to 4, wherein (i), (ii), (iii), and (iv) are operably linked to one or more promoters.

6. 6. The method of claim 5, wherein each of (i), (ii), (iii), and (iv) is operably linked to a separate promoter.

7. 7. The method of claim 6, wherein the promoter operably linked to (i) provides substantially the same level of expression as compared to the promoter operably linked to (ii), (iii), and (iv); or the promoter operably linked to (i) provides at least two-fold higher, or at least three-fold higher, expression as compared to the promoter operably linked to (ii), (iii), and (iv).

8. 8. The method of any one of claims 5 to 7, wherein (i) is operably linked to a promoter and further operably linked to an enhancer, and (ii), (iii), and (iv) are not operably linked to an enhancer.

9. 9. The method of any one of claims 1 to 8, wherein the copy number ratio of (i) to each of (ii), (iii), and (iv) on the one or more vectors is from 1:1 to 5:1, optionally the copy number ratio is 1:1, 2:1 or 3:

1.

10. 10. The method of any one of claims 1 to 9, wherein (ii) comprises E2A and E4; and / or (iv) comprises Rep and Cap.

11. 11. The method of any one of claims 1 to 10, wherein said culturing comprises expressing E1A at a ratio of 1:1 to 5:1 to each of (ii), (iii), and (iv), optionally wherein the ratio is 1:1, 2:1, or 3:

1.

12. The one or more vectors (v) (A) a helper gene selected from E1B, NP1, NS2, or a combination thereof, optionally wherein the E1B is E1B-19k or E1B-55k; and (B) a gene of interest; The method of any one of claims 1 to 11, further comprising one or both of:

13. 13. The method of claim 12, wherein (i) and (v) are on a single vector; or (v) is on a separate vector from (i), (ii), (iii), and (iv).

14. 14. The method of any one of claims 1 to 13, wherein the method produces at least 1.5-fold higher titers of the AAV compared to a method that does not include transfecting an E1 complementing producer cell with an E1A adenoviral helper gene.

15. 1. A method for producing adeno-associated virus (AAV) in an E1 complementation producer cell, comprising: a. the E1 complementation producer cell, i. a first vector comprising E1A operably linked to a first promoter; ii. a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter; iii. a third vector comprising Rep and Cap, both operably linked to a third promoter; and iv. Optionally (A) a helper gene selected from E1B, NP1, NS2, or a combination thereof, which is on a first, second, or third vector, or on a fourth vector; and (B) a gene of interest, the gene being on the first, second, third, or fourth vector, or on a fifth vector. and transfecting the cell with one or both of transfecting, wherein the transfection ratio of (i) to each of (ii) and (iii) is 1:1 to 3:1; b. culturing the transfected E1-complementing producer cells under conditions suitable for producing the AAV; c) purifying said AAV from said cultured E1-complementing producer cells, thereby obtaining said AAV.

16. 1. A method for producing adeno-associated virus (AAV) in an E1 complementation producer cell, comprising: a. the E1 complementation producer cell, i. a first vector comprising E1A operably linked to a first promoter and enhancer; ii. a second vector comprising E2A, E4, and a virus-associated, non-coding RNA (VA RNA), all operably linked to a second promoter; iii. a third vector comprising Rep and Cap, each operably linked to a third promoter; and iv. Optionally (A) a helper gene selected from E1B, NP1, NS2, or a combination thereof, which is on a first, second, or third vector, or on a fourth vector; and (B) a gene of interest, the gene being on the first, second, third, or fourth vector, or on a fifth vector. transfecting the vector with one or both of b. culturing the transfected E1-complementing producer cells under conditions suitable for producing the AAV, wherein E1A is expressed in a ratio of 1:1 to 3:1 to each of E2A, E4, VA RNA, Rep, and Cap; c) purifying said AAV from said cultured E1-complementing producer cells, thereby obtaining said AAV.

17. 1. A method for producing adeno-associated virus (AAV) in an E1 complementation producer cell, comprising: a. the E1 complementation producer cell, i. a first vector comprising E1A, E2A, E4, and VA RNAs, all operably linked to a first promoter; ii. a second vector comprising Rep and Cap operably linked to a second promoter; and iii. Optionally (A) a helper gene selected from E1B, NP1, NS2, or a combination thereof, which is on the first or second vector, or on a third vector; and (B) a gene of interest, the gene being on the first, second, or third vector, or on a fourth vector. and transfecting the cell with one or both of transfecting the vector with a copy number ratio of E1A to each of E2A, E4, VA RNA, Rep, and Cap of 1:1 to 3:1; b. culturing the transfected E1-complementing producer cells under conditions suitable for producing the AAV; c) purifying said AAV from said cultured E1-complementing producer cells, thereby obtaining said AAV.

18. 18. The method of any one of claims 1 to 17, wherein the E1 complementation producer cell is a HEK293 cell, a 911 cell, a pTG6559 cell, a PER.C6 cell, a GH329 cell, a N52.E6 (CAP®) cell, a HeLa-E1 cell, a UR cell, a VLI-293 cell, an Ac51 cell, an Ac139 cell, or a variant or derivative thereof, preferably the E1 complementation producer cell is a HEK293 cell, a PER.C6 cell, or a N52.E6 (CAP®) cell.

19. 19. The method of claim 18, wherein the E1 complementation producer cell is a HEK293 cell, preferably the HEK293 cell is a HEK293S cell, a HEK293T cell, a HEK293F cell, a HEK293FT cell, a HEK293FTM cell, a HEK293SG cell, a HEK293SGGD cell, a HEK293H cell, a HEK293E cell, a HEK293MSR cell, a HEK293A cell, or a combination thereof.

20. 20. The method of claim 19, wherein the HEK293 cells are HEK293T cells.

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