Eukaryotic cells containing adenovirus-associated virus polynucleotides

Abstract

The present invention provides eukaryotic cells, such as mammalian cells, that contain an adeno-associated virus (AAV) polynucleotide, including an AAV capsid protein (Cap), and that can express a polypeptide encoded by the AAV polynucleotide, thereby producing AAV, including recombinant AAV. Eukaryotic cells can also contain adenovirus (Ad) polynucleotides. The present invention also provides methods for expressing AAV polynucleotides, as well as Ad polynucleotides, in eukaryotic cells, such as CHO cells, HEK293, and BHK cells. The present invention further provides other products and methods described herein.

Description

[Technical field]

[0001] This application claims priority to U.S. Application No. 63 / 256,730, filed October 18, 2021, which is incorporated by reference in its entirety.

[0002] The present invention provides eukaryotic cells comprising an adeno-associated virus (AAV) polynucleotide, including an AAV capsid protein (Cap). The cell can express a polypeptide encoded by the AAV polynucleotide, thereby producing AAV, including recombinant AAV. The eukaryotic cell can also comprise an adenovirus (Ad) polynucleotide. The present invention also provides AAV polynucleotides, as well as methods for expressing Ad polynucleotides in eukaryotic cells. The present invention also provides methods for producing recombinant adeno-associated viruses utilizing eukaryotic cells expressing AAV polypeptides and Ad polypeptides encoded by the polynucleotides, as well as recombinant AAV produced by these methods of the present invention. The present invention further provides other products and methods described herein.

[0003] Reference to Electronic Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated by reference herein in its entirety. The .XML copy, created on October 5, 2022, is named "135975-61702.xml" and is 229,879 bytes in size. The Sequence Listing contained in this .XML file is a part of this specification and is incorporated by reference herein in its entirety. [Background technology]

[0004] Adeno-associated viruses (AAV) are non-enveloped, single-stranded DNA viruses used as gene delivery vectors for both research and therapeutics. Weitzman and Linden, Adeno-Associated Virus Biology (chapter 1), Meth. Molec. Biol. 807:1-23 (2011). AAV-based gene transfer vectors have demonstrated promise in human gene therapy based on their safety profile in animal models and the potential to achieve long-term efficacy in humans. Wang et al., Nature, 18:358-78 (2019). A major challenge to advancing AAV-based therapies into clinical development is the difficulty and cost of producing sufficient amounts of AAV through transient methodologies.

[0005] AAV has been produced in HEK293, BHK, human amniotic (e.g., epithelial cells such as HAEpiC), and SF9 lines. However, expression is transient due to the use of plasmid vectors containing the necessary AAV and helper virus genes. For example, recombinant AAV production in HEK293 cells using adenovirus helper gene products utilizes adenovirus E2A, E4, VA RNA, and AAV Rep and Cap, together with AAV inverted terminal repeats (ITRs) flanking the polynucleotide of interest. Reliance on non-integrating plasmid vectors means that the necessary gene products are lost over time and need to be continually re-established.

[0006] The AAV genome contains a capsid gene called "Cap" or "CAP". The natural Cap is translated to produce three size variant structural proteins called VP1 (about 90 kDa), VP2 (about 72 kDa), and VP3 (about 60 kDa) through alternative start codons and transcript splicing. The AAV capsid contains a total of 60 subunits of VP protein. A ratio of 1:1:10 is considered the most typical ratio for VP1:VP2:VP3, with a stoichiometry of 5 VP1 subunits: 5 VP2 subunits: 50 VP3 subunits. However, there can be variation. Worner et al., Nature Communications 12:1642 (2021). AAV polynucleotides and proteins, including CAP, can be selected from any serotype.

[0007] There is therefore a need to develop improved cells and production methods that circumvent the transient nature of non-integrating plasmid vectors. Summary of the Invention

[0008] The present invention provides stable eukaryotic cells, such as mammalian cells (e.g., primate cells, rodent cells, and canine cells) containing integrated AAV and Ad polynucleotides. The protein and VA RNA products of the Ad polynucleotide act in a helper capacity. All AAV and Ad types are acceptable for use according to the present invention. The present invention can advantageously use site-specific integration into the cell genome, which refers to a preselected genomic site for the exogenous DNA to be inserted into the cell genome. Random insertion can be used as well.

[0009] Described herein are polynucleotides, each of which can include (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding an adeno-associated virus (AAV) Cap protein, and (v) a polyadenylation site. For example, the polynucleotide can be in a CHO cell and have (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, and (iv) a polynucleotide encoding an AAV Cap protein that can be operably linked. The polynucleotide can be integrated into a CHO cell genome, such as a CHO chromosome. Alternatively, the polynucleotide can be in a HEK293 cell and have (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, and (iv) a polynucleotide encoding an AAV Cap protein that can be operably linked. The polynucleotide can be integrated into a HEK293 cell genome, such as a HEK293 cell chromosome. Another alternative is a polynucleotide that can be in a BHK cell and has operably linked (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, and (iv) a polynucleotide that encodes an AAV Cap protein. The polynucleotide can be integrated into a BHK cell genome, such as a BHK cell chromosome. In yet another alternative, the polynucleotide can be in a human amniotic cell and has operably linked (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, and (iv) a polynucleotide that encodes an AAV Cap protein. The polynucleotide can be integrated into a human amniotic cell genome, such as a human amniotic cell chromosome. Additionally, the polynucleotide can be integrated into a non-chromosomal location, such as an episome, as known by those skilled in the art.

[0010] The polynucleotide may further comprise an operator. The promoter may be a CMV promoter and the operator may be a Tet operator. The AAV proteins and polynucleotides, including the CAP, may be selected from any serotype. When the Cap is from serotype 5 ("Cap5") and expressed in CHO cells, the polynucleotide allows the production of AAV Cap5 VP2 and AAV Cap5 VP3 proteins, with the amount of VP3 produced being greater than the amount of VP2 produced. The production of VP1 may be at a level less than 1% of VP2 production. The production ratio may vary based on the experimental conditions and analytical techniques.

[0011] Additionally, polynucleotides are described, each of which can include (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding an AAV cap protein, (v) a second internal ribosome entry site, (vi) a second polynucleotide encoding an AAV cap protein, and (vii) a polyadenylation site. For example, the polynucleotide can be in a CHO cell, and the (i) promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding an AAV Cap protein, (v) a second internal ribosome entry site, and (vi) a second polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the CHO cell genome. Alternatively, the polynucleotide can be in a HEK293 cell, and (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding an AAV Cap protein, (v) a second internal ribosome entry site, and (vi) a second polynucleotide encoding an AAV Cap protein are operably linked. The polynucleotide can be integrated into the HEK293 cell genome. In another alternative, the polynucleotide can be in a BHK cell, and (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding an AAV Cap protein, (v) a second internal ribosome entry site, and (vi) a second polynucleotide encoding an AAV Cap protein are operably linked. The polynucleotide can be integrated into the BHK cell genome. In another alternative, the polynucleotide can be in a human amniotic cell, and (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding an AAV Cap protein, (v) a second internal ribosome entry site, and (vi) a second polynucleotide encoding an AAV Cap protein are operably linked. The polynucleotide can be integrated into the human amniotic cell genome.The polynucleotide can further comprise an operator. The promoter can be a CMV promoter and the operator can be a Tet operator. The AAV proteins and polynucleotides including the CAP can be selected from any serotype. When the Cap is from serotype 5 ("Cap5") and expressed in CHO cells, the polynucleotide allows for the production of AAV Cap5 proteins VP1, VP2, and VP3. The amount of VP3 production can be greater than the amount of VP1 production and the amount of VP2 production. The amount of VP2 production can be greater than the amount of VP1 production. The production ratio can vary based on the experimental conditions and the analytical technique.

[0012] Further described are polynucleotides, each of which can include (i) a promoter, (ii) an intron, (iii) a polynucleotide encoding an AAV Cap protein, and (iv) a polyadenylation site, and when the polynucleotide is expressed, allows for the production of an AAV Cap VP1 protein. For example, the polynucleotide can be in a CHO cell, and the (i) promoter, (ii) intron, and (iii) a polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the CHO cell genome. Alternatively, the polynucleotide can be in a HEK293 cell, and the (i) promoter, (ii) intron, and (iii) a polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the HEK293 cell genome. In another alternative, the polynucleotide can be in a BHK cell, and the (i) promoter, (ii) intron, and (iii) a polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the BHK cell genome. In yet another alternative, the polynucleotide can be in a human amniotic cell, and (i) a promoter, (ii) an intron, and (iii) a polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the human amniotic cell genome. The polynucleotide can further comprise an operator. The promoter can be a CMV promoter, and the operator can be a Tet operator.

[0013] Further described are polynucleotides, each of which can include (i) a promoter, (ii) an internal ribosome binding site, (iii) a polynucleotide encoding an AAV Cap protein, and (iv) a polyadenylation site, and when the polynucleotide is expressed, allows for the production of an AAV VP1 Cap protein. The polynucleotide can be in a CHO cell, and the (i) promoter, (ii) an internal ribosome binding site, and the (iii) polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the CHO genome. Alternatively, the polynucleotide can be in a HEK293 cell, and the (i) promoter, (ii) an internal ribosome binding site, and the (iii) polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the HEK293 cell genome. In another alternative, the polynucleotide can be in a BHK cell, and the (i) promoter, (ii) an internal ribosome binding site, and the (iii) polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the BHK cell genome. In yet another alternative, the polynucleotide can be in a human amniotic cell, and the polynucleotide encoding (i) a promoter, (ii) an internal ribosome binding site, and (iii) an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the human amniotic cell genome. The polynucleotide can include an operator. The promoter can be a CMV promoter, and the operator can be a Tet operator.

[0014] Additionally, eukaryotic cells are described in which each cell can contain a polynucleotide comprising: (i) a promoter; (ii) an intron; (iii) a first internal ribosome entry site; (iv) a first polynucleotide encoding an AAV Cap protein; (v) a second internal ribosome entry site; (vi) a second polynucleotide encoding an AAV Cap protein; and (vii) a polyadenylation site. (i) the promoter; (ii) an intron; (iii) the first internal ribosome entry site; (iv) the first polynucleotide encoding an AAV Cap protein; (v) the second internal ribosome entry site; and (vi) the second polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into the cell genome. The cell can be a CHO cell, a HEK293 cell, a BHK cell, a human amniotic cell, or other eukaryotic cell. The cell can further comprise an operator. The promoter can be a CMV promoter, and the operator can be a Tet operator. The cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf6, a polynucleotide encoding VA RNA, and a polynucleotide encoding the AAV ITRs and a protein of interest.

[0015] Also described are eukaryotic cells, each of which comprises (A) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding an AAV Cap protein, and (v) a polyadenylation site, and (B) a second polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a polynucleotide encoding an AAV Cap protein, and (iv) a polyadenylation site. (A) The (i) promoter, (ii) intron, (iii) internal ribosome entry site, and (iv) a polynucleotide encoding an AAV Cap protein of the first polynucleotide can be operably linked, and (B) the (i) promoter, (ii) intron, and (iii) a polynucleotide encoding an AAV Cap protein of the second polynucleotide can be operably linked. The cell can have at least one polynucleotide integrated into the cell genome. The cell can be a CHO cell, a HEK293 cell, a BHK cell, a human amniotic cell, or other eukaryotic cell. The cell can further comprise an operator. The promoter can be a CMV promoter and the operator can be a Tet operator. The cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding E4 or E4 orf6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest.

[0016] Additionally, eukaryotic cells are described, each of which comprises (A) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding an AAV Cap protein, and (v) a polyadenylation site, and (B) a second polynucleotide comprising (i) a promoter, (ii) an internal ribosome entry site, (iii) a polynucleotide encoding an AAV Cap protein, and (iv) a polyadenylation site. The cell can have (A) the polynucleotide encoding (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, and (iv) an AAV Cap protein of the first polynucleotide operably linked to (B) the polynucleotide encoding (i) a promoter, (ii) an internal ribosome entry site, and (iii) an AAV Cap protein of the second polynucleotide operably linked. At least one polynucleotide can be integrated into the cell genome. The cell can be a CHO cell, a HEK293 cell, a BHK cell, a human amniotic cell, or other eukaryotic cell. The cell can further comprise an operator. The promoter can be a CMV promoter and the operator can be a Tet operator. The cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest.

[0017] Also described are cell cultures comprising any of the above cells in any type of medium, including growth medium and maintenance medium. Additionally, methods of producing AAV proteins, including the Cap protein, and methods that can result in the production of recombinant AAV are described.

[0018] A method for producing adeno-associated virus (AAV) Cap protein in cell culture is described, the method comprising the steps of providing a eukaryotic cell, the cell comprising a polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding an AAV Cap protein, (v) a second internal ribosome entry site, (vi) a second polynucleotide encoding an AAV Cap protein, and (vii) a polyadenylation site, and culturing the cell in a culture medium to allow the cell to produce the AAV Cap protein, the polynucleotide allowing the production of AAV Cap proteins VP1, VP2, and VP3. The (i) promoter, (ii) intron, (iii) the first internal ribosome entry site, (iv) the first polynucleotide encoding the AAV Cap protein, (v) the second internal ribosome entry site, and (vi) the second polynucleotide encoding the AAV Cap protein can be operably linked. The polynucleotide can be integrated into the cell genome. The cell can be a CHO cell, a HEK293 cell, a BHK cell, a human amniotic cell, or other eukaryotic cell. The cell can further comprise an operator. The cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest, and the cell can produce recombinant AAV.

[0019] Also described is a method for producing adeno-associated virus (AAV) Cap protein in cell culture, the method comprising the steps of providing a eukaryotic cell, the cell comprising (a) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding an AAV Cap protein, and (v) a polyadenylation site, and (b) a second polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a polynucleotide encoding an AAV Cap protein, and (iv) a polyadenylation site; and culturing the cell in a culture medium to allow the cell to produce the AAV Cap protein, wherein the polynucleotides allow for the production of AAV Cap proteins VP1, VP2, and VP3. (a) the first polynucleotide's (i) promoter, (ii) intron, (iii) internal ribosome entry site, and (iv) polynucleotide encoding an AAV Cap protein can be operably linked, and (b) the second polynucleotide's (i) promoter, (ii) intron, and (iii) polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into a cell genome. The cell can be a CHO cell, a HEK293 cell, a BHK cell, a human amniotic cell, or other eukaryotic cell. The cell can further comprise an operator. The promoter can be a CMV promoter, and the operator can be a Tet operator. The cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest, and the cell can produce a recombinant AAV.

[0020] Also described is a method for producing adeno-associated virus (AAV) Cap protein in cell culture, the method comprising the steps of providing a eukaryotic cell, the cell comprising (a) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding an AAV Cap protein, and (v) a polyadenylation site, and (b) a second polynucleotide comprising (i) a promoter, (ii) an internal ribosome entry site, (iii) a polynucleotide encoding an AAV Cap protein, and (iv) a polyadenylation site; and culturing the cell in a culture medium to allow the cell to produce the AAV Cap protein, wherein the polynucleotides allow for the production of AAV Cap proteins VP1, VP2, and VP3. (a) the first polynucleotide's (i) promoter, (ii) intron, (iii) internal ribosome entry site, and (iv) polynucleotide encoding an AAV Cap protein can be operably linked, and (b) the second polynucleotide's (i) promoter, (ii) internal ribosome entry site, and (iii) polynucleotide encoding an AAV Cap protein can be operably linked. The polynucleotide can be integrated into a cell genome. The cell can be a CHO cell, a HEK293 cell, a BHK cell, a human amniotic cell, or other eukaryotic cell. The cell can further comprise an operator. The promoter can be a CMV promoter, and the operator can be a Tet operator. The cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest, and the cell can produce a recombinant AAV. [Brief description of the drawings]

[0021] [Figure 1] 1 is a schematic diagram of a polynucleotide, including a promoter, an intron, an internal ribosome entry site (IRES), a polynucleotide encoding the AAV Cap protein, and a polyadenylation site (polyA). [Diagram 2] 1 is a schematic diagram of a polynucleotide that includes a promoter, an intron, two internal ribosome entry sites, two polynucleotides encoding the AAV Cap proteins, and a polyadenylation site. [Diagram 3] 1 is a schematic diagram of a polynucleotide, including a promoter, an intron, a polynucleotide encoding the AAV Cap protein, and a polyadenylation site. [Figure 4] 1 is a schematic diagram of a polynucleotide, including a promoter, an internal ribosome entry site, a polynucleotide encoding the AAV Cap protein, and a polyadenylation site. [Diagram 5] 1 is a schematic diagram of a polynucleotide including a promoter with an operator (Op), an intron, an internal ribosome entry site, a polynucleotide encoding the AAV Cap protein, and a polyadenylation site. [Figure 6] 1 is a schematic diagram of a polynucleotide that includes a promoter with an operator (Op), an intron, two internal ribosome entry sites, two polynucleotides encoding AAV Cap proteins, and a polyadenylation site. [Figure 7] 1 is a schematic diagram of a polynucleotide, including a promoter (Op), an intron, a polynucleotide encoding the AAV Cap protein, and a polyadenylation site. [Figure 8] 1 is a schematic diagram of a polynucleotide, including a promoter with an operator (Op), an internal ribosome entry site, a polynucleotide encoding the AAV Cap protein, and a polyadenylation site. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] definition The term "about" in the context of numerical values ​​and ranges refers to a value or range that is approximately or very close to the recited value or range so that the invention can be performed to have the desired rate, amount, degree, increase, decrease, or degree of expression, concentration, or time, as is clear from the teachings contained herein. Thus, the term encompasses values ​​beyond those that simply result from systematic error. For example, "about" can indicate either a value above or below the recited value by a range of approximately + / - 10% or more or less, depending on the ability to perform.

[0024] An "intron" is a portion of DNA located between exons. Introns are removed to form mature messenger RNA. Preferred introns are those that can affect the start point of translation, exemplified by the hCMV-IE intron (human cytomegalovirus immediate early protein) and FMDV intron (foot and mouth disease virus). Globin gene introns have also reportedly been used for expression.

[0025] A "nucleic acid portion" includes any arrangement of single- or double-stranded nucleotide sequences. A nucleic acid portion can include, but is not limited to, polynucleotides, promoters, enhancers, operators, repressors, transcription termination signals, ribosome entry sites, and polyadenylation signals.

[0026] A "DNA cassette" or "cassette" is a type of nucleic acid segment that contains at least a promoter, at least one open reading frame, and optionally, a polyadenylation signal, e.g., an SV40 polyadenylation signal. Other nucleic acid segments, such as an operator, are also optional. Thus, a DNA cassette is a polynucleotide that contains two or more shorter polynucleotides.

[0027] "Operably linked" refers to one or more nucleotide sequences in a functional relationship with one or more other nucleotide sequences. Such a functional relationship can directly or indirectly control, generate, regulate, enhance, promote, permit, attenuate, inhibit, or block an action or activity according to the selected design. Typical include single-stranded or double-stranded nucleic acid portions and can include two or more nucleotide sequences arranged within a given portion such that the sequence(s) can exert at least one functional effect on the other(s). For example, a promoter operably linked to a coding region of a DNA polynucleotide sequence can promote transcription of the coding region. Other elements such as enhancers, operators, repressors, transcription termination signals, ribosome entry sites, and polyadenylation signals can also be operably linked to a polynucleotide of interest to control its expression. The location and spacing to achieve operably linked can be confirmed by approaches available to those skilled in the art, such as screening using Western blots and RT-PCR.

[0028] "Operator" refers to a DNA sequence that is introduced into or near a polynucleotide sequence such that the polynucleotide sequence can be regulated by the interaction of a molecule capable of binding to the operator, resulting in preventing or enabling transcription of the polynucleotide sequence, as the case may be. Those skilled in the art will recognize that an operator must be placed close enough to the promoter to be able to control or affect transcription by the promoter, and can be considered a type of operable linkage. The operator can be placed either downstream or upstream of the promoter. These include, but are not limited to, the operator region of the LexA gene of E. coli, which binds to the LexA peptide and lactose, and the 45 tryptophan operator, which binds to the repressor protein encoded by the Lad and trpR genes of E. coli. Bacteriophage operators from lambda Pi and phage P22 Mnt, as well as Arc. Preferred operators are the Tet (tetracycline) operator and the Arc operator. The operator can have a native sequence or a mutant sequence. For example, mutant sequences of the Tet operator are disclosed in Wissmann et al., Nucleic Acids Res. 14:4253-66 (1986).

[0029] The phrases "percent identity" or "% identical" in their various grammatical forms, when describing a sequence, are meant to include homologous sequences that exhibit the recited identities along the contiguous regions of homology, but the presence of gaps, deletions, or insertions in the compared sequences that do not have a homolog are not taken into account when calculating the percent identity. As used herein, the determination of "percent identity" or "% identical" between homologs does not include the comparison of sequences where the homolog does not have a homologous sequence to compare in an alignment. Thus, "percent identity" and "% identical" do not include penalties for gaps, deletions, and insertions.

[0030] "Homologous sequence" in the context of nucleic acid sequences refers to a sequence that is substantially homologous to a reference nucleic acid sequence. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is a complete (i.e., entire) sequence.

[0031] A "polynucleotide" includes a sequence of covalently linked nucleotides, including RNA and DNA. An oligonucleotide is considered a shorter polynucleotide. A gene is a DNA polynucleotide (polydeoxyribonucleic acid) that ultimately codes for a polypeptide, typically transcribed from DNA and translated from RNA (polyribonucleic acid). A DNA polynucleotide can also code for an RNA polynucleotide that is not translated, but rather functions as an RNA "product". The type of polynucleotide (i.e., DNA or RNA) is clear from the context of the use of the term. A polynucleotide referred to or specified by the polypeptide it encodes refers to and encompasses all suitable sequences according to codon degeneracy. Polynucleotides, including those disclosed herein, include percent identity and homologous sequences, where indicated.

[0032] "Polypeptide" or "peptide" refers to a sequence or sequences of covalently linked amino acids. Polypeptides include natural, semi-synthetic, and synthetic proteins, as well as protein fragments. "Polypeptide" and "protein" can be used interchangeably. An oligopeptide is considered a shorter polypeptide.

[0033] A "protein of interest" or "polypeptide of interest" can have any amino acid sequence and includes any protein, polypeptide, or peptide, as well as derivatives, components, domains, chains, and fragments thereof. Includes, but is not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins, and animal (including human) proteins. Protein types can include, but are not limited to, antibodies, bispecific antibodies, multispecific antibodies, antibody chains (including heavy and light chains), antibody fragments, Fv fragments, Fc fragments, Fc-containing proteins, Fc fusion proteins, receptor Fc fusion proteins, receptors, receptor domains, trap and mini-trap proteins, enzymes, factors, inhibitors, activators, ligands, reporter proteins, selection proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters, and contractile proteins. Also included are derivatives, components, chains, and fragments of the above. The sequences can be natural, semi-synthetic, or synthetic. Proteins of interest and polypeptides of interest are encoded by "genes of interest," which can also be referred to as "polynucleotides of interest." Where multiple genes (the same or different) are integrated, they may be referred to as "first," "second," "third," "fourth," "fifth," "sixth," "seventh," "eighth," "ninth," "tenth," etc., as is clear from the context of usage.

[0034] "Promoter" refers to a DNA sequence that causes transcription of a DNA sequence to which it is operably linked, i.e., linked in such a way as to allow transcription of the nucleotide sequence of interest when appropriate signals are present and repressors are absent. Expression of the polynucleotide of interest can be under the control of any promoter or enhancer element known in the art. Eukaryotic promoters can be operably linked to a TATA box. The TATA box is typically located upstream of the transcription initiation site.

[0035] Useful promoters that may be used include, but are not limited to, the SV40 early promoter region, the SV40E / L (early late) promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequence of the metallothionein gene, the mouse or human cytomegalovirus major immediate early (CMV-MIE) promoter, and other CMV promoters, including the CMVmin promoter. Plant expression vectors containing the nopaline synthase promoter region, the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase; promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: elastase I, insulin, immunoglobulins, mouse mammary tumor virus, albumin, C-fetoprotein, C.1-antitrypsin, 3-globin, and myosino light chain-2. Various forms of the CMV promoter may be used in accordance with the present invention.

[0036] Minimal promoters, such as the CMVmin promoter, can be truncated or core promoters and are preferred for use in regulated expression systems.Minimal promoters and development approaches are widely known and are disclosed, for example, in Saxena et al., Methods Molec.Biol.1651:263-73(2017); Ede et al., ACS Synth Biol.5:395-404(2016); Brown et al., Biotech Bioeng.111:1638-47(2014); Morita et al., Biotechniques 0:1-5(2012); Lagrange et al., Genes Dev.12:34-44(1998). There are many CMVmin promoters reported in the field.

[0037] A "reporter protein" as used herein refers to any protein capable of generating a detectable signal. Reporter proteins typically fluoresce or catalyze a colorimetric or fluorescent reaction and are often referred to as "fluorescent proteins" or "color proteins". However, reporter proteins can also be non-enzymatic and non-fluorescent, so long as they can be detected by another protein or moiety, such as a cell surface protein detected with a fluorescent ligand. Reporter proteins can also be inactive proteins that become functional through interaction with another protein that is fluorescent or catalyzes a reaction. Thus, any suitable reporter protein can be used, as will be appreciated by those of skill in the art. In some embodiments, the reporter protein can be selected from fluorescent proteins, luciferase, alkaline phosphatase, β-galactosidase, β-lactamase, dihydrofolate reductase, ubiquitin, and variants thereof. Fluorescent proteins are useful in some cases for the recognition of gene cassettes that may or may not have been successfully inserted and / or exchanged. Fluid cytometry and fluorescence activated cell sorting are suitable for detection. Examples of fluorescent proteins are well known in the art and include, but are not limited to, Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), and far-red fluorescent protein (e.g., mKate, mKate2, mPlum, mRaspberry, or E2-crimson. See, e.g., U.S. Pat. No. 9,816,110. Reporter proteins are encoded by polynucleotides and are referred to herein as "reporter genes" or "reporter protein genes." A reporter can be considered a type of marker. "Color" or "fluorescence" can also be used in their various grammatical forms to refer more specifically to a reporter protein or gene.

[0038] A "repressor protein", also referred to as a "repressor", is a protein capable of binding to DNA for repressor transcription. Repressors are of eukaryotic and prokaryotic origin. Prokaryotic repressors are preferred. Examples of repressor families include the TetR, LysR, LacI, ArsR, IcIR, MerR, AsnC, MarR, DeoR, GntR, and Crp families. Repressor proteins in the TetR family include ArcR, ActII, AmeR, AmrR, ArpR, BpeR, EnvR, EthR, HemR, HydR, IfeR, LanK, LfrR, LmrA, MtrR, Pip, PqrA, QacR, RifQ, RmrR, SimReg2, SmeT, SrpR, TcmR, TetR, TtgR, TrgW, UrdK, VarR YdeS, ArpA., BarA, Aur1B, CalR1, CprB, FarA, JadR*, JadR2, MphB, NonG, PhlF, TylQ, VanT, TarA, TylP, BM1P1, Bm3R1, ButR, CampR, CamR, DhaR, KstR, LexA, AcnR, PaaRR, PsbI, Th1R, U idR, YDH1, BetI, McbR, MphR, PhaD, Q9ZF45, TtK, Yhgd, YixD, CasR, IcaR, LitR, LuxR, LuxT, O Examples include paR, Orf2, SmcR, HapR, Ef0113, HlyIIR, BarB, ScbR, MmfR, AmtR, PsrA, and YjdC proteins. See Ramos et al., Microbiol. Mol. Biol. Rev., 69:326-56 (2005). Still other repressors include PurR, LacR, MetJ, and PadR, and the repressor proteins are encoded by genes referred to as "repressor genes" or "repressor protein genes."

[0039] "Selectable" or "selectable" marker proteins include proteins that confer a particular trait, including but not limited to drug resistance or other selective advantage. A selectable marker can result in a cell that has received a selectable marker gene that is resistant to a particular toxin, drug, antibiotic, or other compound, allowing the cell to produce the protein and grow in the presence of the toxin, drug, antibiotic, or other compound, and is often referred to as a "positive selectable marker." Suitable examples of antibiotic resistance markers include, but are not limited to, proteins that confer resistance to various antibiotics, such as kanamycin, spectinomycin, neomycin, gentamicin (G418), ampicillin, tetracycline, chloramphenicol, puromycin, hygromycin, zeocin, and / or blasticidin. There are other selectable markers, often referred to as "negative selectable markers," that cause the cell to stop growing, stop producing protein, and / or are lethal to the cell in the presence of the negative selectable marker protein. Thymidine kinase and certain fusion proteins can function as negative selectable markers, including but not limited to GyrB-PKR. See White et al., Biotechniques, 50:303-309 (May 2011). Selectable marker proteins and corresponding genes can be generally referred to as first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as will be clear from the context of use.

[0040] All numerical limits and ranges set forth herein include all numbers or values ​​surrounding or between the numbers of the range or limit. The ranges and limits set forth herein expressly represent and set forth all integer, decimal, and fractional values ​​encompassed by the range or limit.

[0041] The invention provides cells containing AAV polynucleotides and, optionally, Ad polynucleotide sequences to allow for the production of recombinant AAV containing a polynucleotide of interest, such as a gene or other sequence encoding a polypeptide of interest. The AAV and Ad polynucleotides provide the structural and helper products required for AAV production.

[0042] The AAV polynucleotide, and optionally the Ad polynucleotide, can be integrated, for example, using recombinase-mediated cassette exchange (RMCE). "Stable" in the context of cellular integration refers to a polynucleotide of interest, such as a gene, that is introduced into the genome of a cell and can be passed on to subsequent generations of cells, thereby providing a cell line that is genetically homogeneous over a period of time.

[0043] Cells suitable for use in the present invention can be readily selected by one of skill in the art. In some embodiments, the cell line is a eukaryotic cell line, such as a yeast cell line, an insect cell line (e.g., Sf9 and Sf21 cells), or a mammalian cell line. Preferred mammalian cells include primate cells (including human), canine cells, and rodent cells. The cells can be primary or immortalized cells. Suitable cells include Vero cells, COS cells, HEK293 cells, HeLa cells, CHO cells, BHK cells, MDCK cells, amniotic cells (human), embryonic cells, Immortalized human retinal cells transfected with adenovirus genes, including but not limited to PER.C6 cells or NSO cells; The cell may be selected from cell lines transfected with viral genes, such as AD5 E1. In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell line. Some examples of CHO cells include, but are not limited to, CHO-ori, CHO-K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and mutants and variants thereof. In other embodiments, the cell is a HEK293 cell. Some examples of HEK293 cells include, but are not limited to, HEK293, HEK293A, HEK293E, HEK293FTM, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T, and mutants and variants thereof.

[0044] In hamster cells such as CHO and BHK, integration can be achieved by the inventions disclosed in U.S. Patent Nos. 7,771,997 ("Stable Site 1") and 9,816,110 ("Stable Site 2"), which are incorporated herein by reference, including sequence information. Regeneron offers a line of products and services called EESYR®. CHO cells with sequences integrated into Stable Site 1 and Stable Site 2 are disclosed in US2019 / 0233544A1, which are incorporated herein by reference, including sequence information. The sequences shown in these patents and in Examples 14 and 15 can be used in accordance with the inventions described and illustrated herein. Additionally, the AAVS1-like region and COSMC locus in hamster cells can be used in accordance with the present invention.

[0045] When using human cells, integration can be performed at the adeno-associated virus integration site 1 (AAVS1). See Lou et al., Human Gene Therapy Methods, 28:124-38 (2017); Liu et al., BMC Research Note, 7:626 (2014). AAVS1 is reported to be located on chromosome 19. Other integration sites in human cells, such as CCR5 and hROSA26, can be used as well.

[0046] Modification of the cell genome can be performed by known approaches such as Cre / Lox, Flp / Frt, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, zinc finger nucleases (ZFNs), ZFN dimers, or RNA-guided DNA endonuclease systems such as CRISPR / Cas9. See U.S. Patent No. 9,816,110 at cols. 17-18. Integration can also be performed using Bxb1 integrase in human, mouse, and rat cells. Russell et al., Biotechniques 40:460-64 (2006).

[0047] To maximize stability and efficiency and to facilitate integration and control of the present invention, Stable Integration Sites (SIS) can be created using genomic safe harbors and the like in a wide variety of cell types and cell lines following the teachings of US63 / 256,675. The descriptions (including examples) and figures providing methods and cells resulting from the methods of US63 / 256,675 are incorporated herein by reference.

[0048] In producing recombinant AAV, the invention provides for the incorporation of the AAV Cap to produce size variants VP1 (approximately 90 kDa), VP2 (approximately 72 kDa), and VP3 (approximately 60 kDa). The variants differ at their N-termini.

[0049] Typically, a recombinant AAV contains a gene of interest (GOI) flanked by AAV ITRs (inverted terminal repeats). For the production of recombinant AAV, seven additional polynucleotides are typically used for production: adenovirus E1A, E1B, E4, E2A, VA RNA, and AAV Rep and Cap.

[0050] Figures 1-4 show constructs that allow for constitutive expression of the Cap protein. Figures 5-8 show constructs that allow for controlled expression of the Cap protein by including an operator downstream of the promoter. A preferred operator is the tetracycline operator (TetO), which binds to the tetracycline repressor (TetR). Tetracycline, doxycycline, and their derivatives can bind to TetR such that TetR no longer binds to TetO, thus allowing transcription. An example of a CMV promoter and TetO is shown in Example 13. EXAMPLES

[0051] The present invention is further described by the following examples which illustrate various aspects of the invention but are not intended to be limiting in any way.

[0052] Example 1 - CHO cells One or more Cap-containing polynucleotides according to Figures 1-8 are stably inserted into the CHO genome. A preferred promoter is the hCMV-IE promoter, and optionally, a tet operator can be operably linked to the promoter for expression control. Optionally, an intron can be located 3' of the promoter. A preferred intron is the hCMV-IE intron. AAV Cap, Rep, and ITRs can be obtained from any AAV serotype. Preferred AAV serotypes are AAV2 and AAV5. AAV polynucleotide sequences are shown in Example 11. Promoter, operator, IRES, and intron sequences are shown in Example 13.

[0053] The AAV ITRs, AAV Rep, and Ad E1A, E1B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf6)), and VA RNAs flanking the gene of interest can be randomly integrated, site-specifically integrated, or remain on the plasmid. Adenovirus polynucleotide sequences are available and are exemplified in Example 12. Adenovirus (Ad) proteins and polynucleotides can be selected from any serotype.

[0054] Example 2 - HEK293 cells One or more Cap-containing polynucleotides according to Figures 1-8 are stably inserted into the HEK293 genome. A preferred promoter is the hCMV-IE promoter, and optionally, a tet operator can be operably linked to the promoter for expression control. Optionally, an intron can be located 3' of the promoter. A preferred intron is the hCMV-IE intron. AAV Cap, Rep, and ITRs can be obtained from any AAV serotype. AAV2 and AAV5 AAV serotypes are preferred. AAV polynucleotide sequences are shown in Example 11.

[0055] The AAV ITRs and Rep, and the Ad E1A, E1B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf6)), and VA RNAs can be randomly integrated, site-specifically integrated, or remain on the plasmid. Adenovirus polynucleotide sequences are available and are exemplified in Example 12.

[0056] Example 3 - BHK Cells BHK cells are fibroblasts from baby hamster kidney. There are adherent BHK strains and BHK strains that can grow in suspension. Wentz and Schuegerl, Enzyme Microbial Tech. 14:68-75 (1992).

[0057] One or more Cap-containing polynucleotides according to Figures 1-8 are stably inserted into the BHK genome. A preferred promoter is the hCMV-IE promoter, and optionally, a tet operator can be operably linked to the promoter for expression control. Optionally, an intron can be located 3' of the promoter. A preferred intron is the hCMV-IE intron. AAV Cap, Rep, and ITRs can be obtained from any AAV serotype. AAV2 and AAV5 AAV serotypes are preferred. AAV polynucleotide sequences are shown in Example 11.

[0058] The AAV ITRs and Rep, and Ad E1A, E1B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf6)), and VA RNAs can be randomly integrated, site-specifically integrated, or remain on the plasmid. Adenovirus polynucleotide sequences are available and are exemplified in Example 12.

[0059] Example 4 - Intron IRES CAP An embodiment of this construct is shown in Figures 1 (constitutive) and 5 (controllable). In cells, both embodiments can produce mainly VP2 and VP3, with more VP3 produced than VP2. In experiments with CHO cells using Cap5, an average ratio of about 1:5.7 of VP3:VP2 was observed by performing densitometric analysis on Western blots. Some VP1 can also be produced, but the levels observed were typically less than 1% of the levels of VP2 produced. Thus, this construct can be used with constructs that produce mainly VP1. See Examples 6 and 7. The production ratio can vary based on the experimental conditions and the analytical technique. A preferred IRES is the Encephalomyocarditis Virus (also called "EMCV" or "ECMV") IRES.

[0060] Example 5 - Intron IRES CAP An embodiment of this construct is shown in Figures 2 (constitutive) and 6 (controllable). This construct contains two IRES polynucleotides and two Cap polynucleotides. In cells, both embodiments can produce VP1, VP2, and VP3. The amount of VP3 production can be greater than the amount of VP1 production and the amount of VP2 production. The amount of VP2 production can be greater than the amount of VP1 production. In experiments using CHO cells containing Cap5, an average ratio of VP1:VP2:VP3 of about 1:2:9.3 was observed by performing densitometric analysis on Western blots. The production ratio can vary based on the experimental conditions and analysis technique. A preferred IRES is the Encephalomyocarditis Virus (referred to as "EMCV" or "ECMV") IRES.

[0061] Example 6 - Intron CAP An embodiment of this construct is shown in Figures 3 (constitutive) and 7 (controllable). This construct contains an intron and Cap polynucleotide, produces primarily VP1, and can be used with the construct of Example 4 to produce VP1, VP2, and VP3.

[0062] Example 7 - IRES CAP An embodiment of this construct is shown in Figures 4 (constitutive) and 8 (controllable). This construct contains an intron and Cap polynucleotide and produces primarily VP1, and can be used with the construct of Example 4 to produce VP1, VP2, and VP3. A preferred IRES is the encephalomyocarditis virus (also referred to as "EMCV" or "ECMV") IRES.

[0063] Example 8 - CHO cells containing Cap constructs The CHO cells of Example 1 can contain the intronic IRES CAP IRES CAP polynucleotide of Example 5 (FIGS. 2 or 6). In experiments, CHO cells containing Cap5 expressed VP1:VP2:VP3 in a ratio of approximately 1:2:9.3, as observed by densitometric analysis on Western blots.

[0064] Alternatively, the CHO cells of Example 1 can contain the CAP polynucleotides of Example 4 (Figures 1 or 5) and Example 6 (Figures 3 or 7) or 6 (Figures 4 and 8). Such cells will express VP1, VP2, and VP3, but perhaps in a different ratio than Example 5.

[0065] Example 9 - HEK293 cells containing Cap constructs The HEK293 cells of Example 2 can contain the intronic IRES CAP IRES CAP polynucleotide of Example 5 (FIGS. 2 or 6).

[0066] Alternatively, the HEK293 cells of Example 2 can contain the CAP polynucleotides of Example 4 (Figures 1 or 5) and Example 6 (Figures 3 or 7) or 7 (Figures 4 and 8). Such cells will express VP1, VP2, and VP3, but perhaps in a different ratio than Example 5.

[0067] Example 10 - BHK cells containing Cap constructs The BHK cells of Example 3 can contain the intronic IRES CAP IRES CAP polynucleotide of Example 5 (FIGS. 2 or 6).

[0068] Alternatively, the BHK cells of Example 3 can contain the CAP polynucleotides of Example 4 (Figures 1 or 5) and Example 6 (Figures 3 or 7) or 7 (Figures 4 and 8). Such cells would express VP1, VP2, and VP3, but perhaps in a different ratio than Example 5.

[0069] Example 11 - AAV Polynucleotide Sequences AAV Rep, Cap, and ITR sequences are known in the art. The present invention is compatible with all AAV serotypes. AAV sequences from various AAV serotypes are shown below. Many of these sequences are available from the National Center for Biotechnology Information (NCBI). AAV-1 Whole genome:NC_002077 CapVP1: (SEQ ID NO: 1) Rep78: (SEQ ID NO: 2) AAV-2 Whole genome:NC_001401 Rep78: (SEQ ID NO: 3) Rep52: (SEQ ID NO: 4) CapVP1: (SEQ ID NO: 5) CapVP2: (SEQ ID NO: 6) CapVP3: (SEQ ID NO: 7) CapAAP: (Accession No. 8) CTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGA AAV-3 Full genome: NC_001729 Rep78: (Accession No. 9) CapVP1: (SEQ ID NO: 10) AAV-4 Whole genome:NC_001829 Rep78: (SEQ ID NO: 11) CapVP1: (SEQ ID NO: 12) AAV-5 Whole genome:NC_006152 Rep78: (SEQ ID NO: 13) CapVP1: (SEQ ID NO: 14) AAV-6 Whole genome:AF028704 Rep78: (SEQ ID NO: 15) CapVP1: (SEQ ID NO: 16) AAV-7 Whole genome:NC_006260 Rep78: (SEQ ID NO: 17) CapVP1: (SEQ ID NO: 18) AAV-8 Whole genome:NC_006261 Rep78: (SEQ ID NO: 19) CapVP1: (SEQ ID NO: 20) AAV-9 Cap only AY530579 CapVP1: (SEQ ID NO: 21) AAV-10 Partial genome:AY631965 Rep78: (SEQ ID NO: 22) CapVP1: (SEQ ID NO: 23) AAV-11 Partial genome:AY631966 Rep78: (SEQ ID NO: 24) CapVP1: (SEQ ID NO: 25) AAV-12 Partial genome: DQ813647 Rep78: (SEQ ID NO: 26) CapVP1: (SEQ ID NO: 27) AAV-13 Partial genome: EU285562 Rep78: (SEQ ID NO: 28) CapVP1: (SEQ ID NO: 29) ITR sequence (SEQ ID NO:30) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Rep2 sequence - includes Rep78 and Rep52 (start codon is underlined) (SEQ ID NO:31) JPEG2024537896000001.jpg104169Cap2 sequence - consecutively containing VP1, VP2, AAP, and VP3 (start codon is underlined) (SEQ ID NO: 32) JPEG2024537896000002.jpg48169 JPEG2024537896000003.jpg78169Cap5 sequence - consecutively containing VP1, VP2, AAP, VP3 (start codon is underlined) (SEQ ID NO:33) JPEG2024537896000004.jpg119169 Example 12 - Adenovirus polynucleotide sequence The adenovirus (Ad) polynucleotide may be selected from any serotype, with representative polynucleotides exemplified below. E2A full sequence (SEQ ID NO:34) GAGTGTACAAATAAAAACATTTGCCTTTATTGAAAGTGTCTCCTAGTACATTATTTTTACATGTTTTTCAAGTGACAAAAAGAAGTGGCGCTCCTAATCTGCGCACTGTGGCTGCGGAAGTAGGGCGAGTGGCGCTCAGGAAGCTGTAGAGCTGTTCCTGGTTGCGACGCAGGGTGGGCTGTACCTGGGGACTGTTAAGCATGGAGTTGGGTACC E2A ORF sequence (SEQ ID NO:35) E4 full sequence (SEQ ID NO:36) E4 Orf6 sequence (SEQ ID NO: 37) ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATGTAG VA sequence (underlined in VA transcripts I and II) (SEQ ID NO: 38) JPEG2024537896000005.jpg43169The sequences of E1A and E1B are both contained in accession AY339865.1. Ad5 E1A Two proteins can be transcribed, a 32 kDa protein (first accession number) and a 27 kDa protein (second accession number), both of which are splice variants from the transcript. Registration 1:AAQ19284.1 Registration 2:AAQ19285.1 (SEQ ID NO:39) ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAA (SEQ ID NO: 40) ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAA Ad5 E1B_19K Registration: AAQ19286.1 (SEQ ID NO: 41) ATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGATAAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGA Ad5 E1B_55K Registration: AAQ19287.1 (SEQ ID NO: 42) The sequences of E2A and E4A are both contained in accession MN088492. Ad5 E2A orf: Registration: QHX41645.1 (SEQ ID NO:43) Ad5 E4A: Two proteins are present in this ORF. The first is a splice variant contained within the ORF. The second is an unspliced ​​transcript present in the ORF. Accession 1: QHX41659.1 Registration 2: QHX41660.1 (SEQ ID NO:44) ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAGTTGGACAGCAGC CTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTGCCCGGGGAGTTTATTAATATCACTGATGAGCGTTTGGCTCGACAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATGCTTTTTAAGGCCAGCCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGAATACTAGGGTTCTGTGA (SEQ ID NO:45) ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATGTAG Ad5 VA: Registration: AF369965.1 (SEQ ID NO: 46) TCGATGTAGGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGTCTGCCGCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGGATGCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTGCAAGCGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCCGAGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTACAGGGAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCGGCCAGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCAAAAGGAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGGTAACCGCATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCGCCATGATACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCTCCTTTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGCGTCCCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTTCCGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGGATTACG Example 13 - Promoter, Operator, IRES, and Intron Sequences CMV promoter (SEQ ID NO: 47) TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT AGTAACGCCAATGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTCGACGTTTAGTGAACCG 2x Tet operator sequence (SEQ ID NO:48) TCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGA hCMV intron sequence (SEQ ID NO:49) GTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCTTTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGCCCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGTCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTCCAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAG ECMV IRES sequence (SEQ ID NO: 50) CCCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATTGCTCGAATCACC FMDV IRES (SEQ ID NO: 51) AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGTGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTG CAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTACAATTAATGACCCT Example 14 - CHO and Mouse Stable Site 1 Sequences - U.S. Patent No. 7,771,997 211>6473 <212> DNA <213> Cricetulus griseus <400> 1 (SEQ ID NO:52) JPEG2024537896000006.jpg55169 JPEG2024537896000007.jpg237169 JPEG2024537896000008.jpg150169 <211> 7045 <212> DNA <213> Cricetulus griseus <400> 2 (SEQ ID NO:53) JPEG2024537896000009.jpg48169 JPEG2024537896000010.jpg235169 JPEG2024537896000011.jpg200169 <211> 6473 <212> DNA <213> Cricetulus griseus <400> 3 (SEQ ID NO:54) JPEG2024537896000012.jpg195169 JPEG2024537896000013.jpg234169 JPEG2024537896000014.jpg10169 <211> 7045 <212> DNA <213> Cricetulus griseus <400> 4 (SEQ ID NO:55) JPEG2024537896000015.jpg183169 JPEG2024537896000016.jpg235169 JPEG2024537896000017.jpg62169 <211> 13515 <212> DNA <213> Cricetulus griseus <400> 5 (SEQ ID NO:56) JPEG2024537896000018.jpg136169 JPEG2024537896000019.jpg235169 JPEG2024537896000020.jpg236169 JPEG2024537896000021.jpg236169 JPEG2024537896000022.jpg78169 <211> 14553 <212> DNA <213> Mus musculus <400> 6 (SEQ ID NO:57) JPEG2024537896000023.jpg124169 JPEG2024537896000024.jpg234169 JPEG2024537896000025.jpg235169 JPEG2024537896000026.jpg236169 JPEG2024537896000027.jpg158169 Example 15 - CHO Stable Site 2 Sequence - U.S. Patent No. 9,816,110 <211> 4001 <212> DNA <213> Cricetulus griseus <400> 1 (SEQ ID NO:58) JPEG2024537896000028.jpg22169 JPEG2024537896000029.jpg233169 JPEG2024537896000030.jpg18169 <211> 14931 <212> DNA <213> Cricetulus griseus <220> <221> misc_feature <222> (2176)..(2239) <223> n is a, c, g, t, or a missing nucleotide <400> 4 (SEQ ID NO:59) JPEG2024537896000031.jpg155169 JPEG2024537896000032.jpg236169 JPEG2024537896000033.jpg235169 JPEG2024537896000034.jpg234169 JPEG2024537896000035.jpg151169

[0070] It should be understood that the description, specific examples, and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the invention. Various changes and modifications within the invention, including combining the embodiments in whole or in part, will become apparent to those skilled in the art from the discussion, disclosure, and data contained herein and are therefore considered part of the invention.

Claims

1. 1. A polynucleotide comprising: (i) a promoter; (ii) an intron; (iii) a first internal ribosome entry site; (iv) a first AAV Cap gene; (v) a second internal ribosome entry site; (vi) a second AAV Cap gene; and (vii) a polyadenylation site.

2. 2. The polynucleotide of claim 1, wherein the polynucleotide is integrated into a CHO cell or HEK293 cell genome and (i) to (vii) are operably linked.

3. The polynucleotide of claim 1 further comprising an operator.

4. The polynucleotide of claim 3 , wherein the promoter is a CMV promoter and the operator is a Tet operator.

5. A polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an AAV Cap gene, and (iv) a polyadenylation site, wherein the polynucleotide, when expressed, enables the production of an AAV Cap VP1 protein.

6. 6. The polynucleotide of claim 5, wherein the polynucleotide is integrated into the HEK293 cell genome or the CHO cell genome.

7. The polynucleotide of claim 5 further comprising an operator.

8. The polynucleotide of claim 7, wherein the promoter is a CMV promoter and the operator is a Tet operator.

9. 1. A eukaryotic cell comprising a polynucleotide comprising: (i) a promoter; (ii) an intron; (iii) a first internal ribosome entry site; (iv) a first AAV Cap gene; (v) a second internal ribosome entry site; (vi) a second AAV Cap gene; and (vii) a polyadenylation site.

10. A eukaryotic cell as described in claim 9, wherein (i) to (vii) are operably linked.

11. The eukaryotic cell of claim 10, wherein the polynucleotide is integrated into the CHO or HEK293 cell genome.

12. A eukaryotic cell as described in claim 9, wherein the polynucleotide further comprises an operator.

13. The eukaryotic cell of claim 12 , wherein the promoter is a CMV promoter and the operator is a Tet operator.

14. a polynucleotide encoding AAV Rep; a polynucleotide encoding Ad E1A; a polynucleotide encoding Ad E1B; a polynucleotide encoding Ad E2A or E2A orf; a polynucleotide encoding Ad E4 or E4 orf6; a polynucleotide encoding a VA RNA; 10. The eukaryotic cell of claim 9, further comprising AAV ITRs and a polynucleotide encoding a protein of interest.

15. A eukaryotic cell, A eukaryotic cell comprising a polynucleotide comprising: (i) a promoter; (ii) an intron; (iii) an AAV Cap gene; and (iv) a polyadenylation site.

16. A eukaryotic cell as described in claim 15, wherein (i) to (iv) are operably linked.

17. A eukaryotic cell as described in claim 16, wherein the polynucleotide is integrated into a CHO or HEK293 cell genome.

18. The eukaryotic cell of claim 15 , wherein the polynucleotide further comprises an operator.

19. 19. The eukaryotic cell of claim 18, wherein the promoter is a CMV promoter and the operator is a Tet operator.

20. a polynucleotide encoding AAV Rep; a polynucleotide encoding Ad E1A; a polynucleotide encoding Ad E1B; a polynucleotide encoding Ad E2A or E2A orf; a polynucleotide encoding Ad E4 or E4 orf6; a polynucleotide encoding a VA RNA; 16. The eukaryotic cell of claim 15, further comprising AAV ITRs and a polynucleotide encoding a protein of interest.

21. 1. A method for producing adeno-associated virus (AAV) Cap protein in cell culture, said method comprising: providing a eukaryotic cell, the cell containing a polynucleotide comprising: (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first AAV Cap gene, (v) a second internal ribosome entry site, (vi) a second AAV Cap gene, and (vii) a polyadenylation site; Culturing the cells in a culture medium to allow the cells to produce the AAV Cap protein.

22. The method of claim 21, wherein (i) to (vii) are operably linked.

23. 23. The method of claim 22, wherein the polynucleotide is integrated into the CHO or HEK293 cell genome.

24. 22. The method of claim 21, wherein the polynucleotide contained in the cell further comprises an operator.

25. 25. The method of claim 24, wherein the promoter is a CMV promoter and the operator is a Tet operator.

26. The eukaryotic cell a polynucleotide encoding AAV Rep; a polynucleotide encoding Ad E1A; a polynucleotide encoding Ad E1B; a polynucleotide encoding Ad E2A or E2A orf; a polynucleotide encoding E4 or E4 orf6; a polynucleotide encoding a VA RNA; 22. The method of claim 21, further comprising AAV ITRs and a polynucleotide encoding a protein of interest, wherein the cell is capable of producing recombinant AAV.

27. 1. A method for producing adeno-associated virus (AAV) Cap protein in cell culture, said method comprising: Providing a eukaryotic cell, the cell comprising: providing a polynucleotide comprising: (i) a promoter; (ii) an intron; (iii) an AAV Cap gene; and (iv) a polyadenylation site; and culturing the cells in a culture medium to allow the cells to produce the AAV Cap protein.

28. The method of claim 27, wherein (i) to (iv) are operably linked.

29. 28. The method of claim 27, wherein the polynucleotide is integrated into the CHO or HEK293 cell genome.

30. 28. The method of claim 27, wherein the polynucleotide in the cell further comprises an operator.

31. 31. The method of claim 30, wherein the promoter is a CMV promoter and the operator is a Tet operator.

32. The eukaryotic cell a polynucleotide encoding AAV Rep; a polynucleotide encoding Ad E1A; a polynucleotide encoding Ad E1B; a polynucleotide encoding Ad E2A or E2A orf; a polynucleotide encoding Ad E4 or E4 orf6; a polynucleotide encoding a VA RNA; 28. The method of claim 27, further comprising AAV ITRs and a polynucleotide encoding a protein of interest, wherein the cell is capable of producing recombinant AAV.