Adeno-associated virus (AAV) production cell lines

Abstract

The present disclosure relates to nucleic acids encoding accessory genes and adeno-associated virus (AAV) genes under the control of inducible promoters. The present disclosure also relates to mammalian cell lines for producing AAV and methods of producing AAV using the mammalian cells.

Description

Technical Field

[0001] This disclosure provides mammalian cell lines for the production of adeno-associated virus (AAV). The cells suitably comprise isolated nucleic acids encoding a helper gene and an AAV gene, controlled by an inducible promoter. This disclosure also relates to methods for producing AAV using the mammalian cells and nucleic acids described herein. Background Technology

[0002] Recombinant adeno-associated virus (rAAV) has become an important gene therapy vector in widespread clinical applications due to its high safety profile and powerful efficacy in delivering functional genes to multiple tissues and generating long-term therapeutic benefits. Several AAV-based gene therapies have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). As of February 2022, 25 viral vector therapies were in late-stage development, and approximately 120 were in Phase II trials; the number of approved therapies is expected to only increase.

[0003] However, current AAV manufacturing processes face challenges including low productivity and scalability, poor batch-to-batch reproducibility, high costs, and complex supply chain issues, which significantly limit the accessibility of this gene delivery platform for treating a variety of human diseases. Furthermore, constitutive expression of both the helper gene and the Rep gene required for AAV production can be cytotoxic. Stable AAV production cell lines (PCLs) and vectors designed to tightly control the expression of the helper and Rep genes can overcome these limitations and support large-scale, current Good Manufacturing Practice (cGMP) manufacturing of AAV gene therapies. Summary of the Invention

[0004] In some embodiments, this disclosure relates to an isolated nucleic acid molecule operable to include: a first inducible promoter having two tetracycline operon sequences (TetO2 sequences); an E2A gene controlled by the first inducible promoter; a virus-associated (VA) noncoding RNA controlled by a second inducible promoter; a third inducible promoter having the TetO2 sequence; an E4 gene controlled by the third inducible promoter; and an antibiotic resistance gene.

[0005] In another embodiment, this disclosure provides an isolated nucleic acid molecule operatively comprising: a first inducible promoter having a TetO2 sequence; an E2A gene controlled by the first inducible promoter; a virus-associated (VA) noncoding RNA controlled by a second inducible promoter; a third inducible promoter having a TetO2 sequence; an E4 gene controlled by the third inducible promoter; an expression cassette of a protein ortholog and peptide tag encoding VA RNA controlled by a repressive promoter, the repressive promoter comprising a human cytomegalovirus (CMV) promoter and a TetO2 sequence; a termination sequence; and an antibiotic resistance gene.

[0006] In other alternative embodiments, this disclosure relates to an isolated nucleic acid molecule operatively comprising a plasmid encoding: a first inducible promoter having a TetO2 sequence; a Rep78 gene containing a silenced p19 promoter located within the coding region of the Rep78 gene, wherein the silenced p19 promoter contains a mutation at the SP1, TATA-1, and / or TATA-2 sites; a second inducible promoter and the TetO2 sequence; a Rep52 gene controlled by the second inducible promoter; a termination sequence; and an antibiotic resistance gene.

[0007] In other embodiments, this disclosure relates to a mammalian cell for producing adeno-associated virus (AAV) comprising any of the isolated nucleic acid molecules disclosed herein integrated into its genome. In some embodiments, the mammalian cell further comprises a nucleic acid encoding a transcriptional repression domain co-located with a nucleic acid encoding a tetracycline repressor protein. In some embodiments, the mammalian cell comprises a nucleic acid molecule encoding a target gene.

[0008] In other additional embodiments, this disclosure provides a method for producing adeno-associated virus (AAV), the method comprising inducing the production of a genome-integrated nucleic acid sequence of any of the mammalian cells disclosed herein, culturing the mammalian cells, and harvesting the AAV. Attached Figure Description

[0009] Figure 1 The diagram shows a Tet-On inducible system that utilizes tetracycline repressor proteins to both inhibit and activate protein expression.

[0010] Figure 2A and Figure 2B Exemplary TetR and TetR-KRAB fusion systems are shown.

[0011] Figures 3A-3I An exemplary nucleic acid molecule for generating an accessory gene is shown according to an embodiment of this document.

[0012] Figure 4A and Figure 4B An exemplary nucleic acid molecule for generating an accessory gene is shown according to an embodiment of this document.

[0013] Figures 5A-5P An exemplary nucleic acid molecule for generating the Rep gene according to the embodiments described herein is shown.

[0014] Figures 6A-6E Exemplary nucleic acid molecules for generating the Cap gene and the target gene according to the embodiments described herein are shown.

[0015] Figure 7 The results show the AAV titers using the helper gene, Rep, and Cap-GOI vector according to the implementation scheme described herein.

[0016] Figure 8 Western blot images showing the expression of accessory proteins, Rep proteins, and Cap proteins according to the embodiments described herein.

[0017] Figure 9 The results show the production of AAV in a 3-liter single-use bioreactor according to the embodiment described herein.

[0018] Figure 10 The comparison results of AAV production by PCL clones are shown.

[0019] Figure 11 The results of cell-based infectivity and potency assays of AAV produced according to the embodiments described herein are shown. Detailed Implementation

[0020] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. In the claims and / or specification, when used in conjunction with the term “comprising / including,” the word “a / an” may mean “a / an,” but also aligns with the meanings of “one or more / a combination of,” “at least one / at least one,” and “one or more / a type or more.”

[0021] Throughout this application, the term "about" is used to indicate values ​​that include inherent error variations in the methods / apparatus used to determine those values. Generally, depending on the circumstances, the term is intended to cover variability of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0022] The use of the term "or" in the claims is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that alternatives are mutually exclusive, although this disclosure supports the definition of referring only to alternatives and "and / or".

[0023] As used in this specification and claims, the terms “comprising” (and any forms thereof, such as “comprise” and “comprises”), “having” (and any forms thereof, such as “have” and “has”), “including” (and any forms thereof, such as “includes” and “include”), or “containing” (and any forms thereof, such as “contains” and “contain”) are inclusive or open-ended and do not exclude other unlisted elements or method steps. It is contemplated that any embodiments discussed in this specification may be implemented with respect to any method, system, host cell, expression vector, and / or composition of the present invention. Furthermore, the compositions, systems, cells, and / or nucleic acids of the present invention can be used to implement any of the methods described herein.

[0024] The use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) indicates that the specific term referenced is a representative example and implementation of this disclosure, and unless otherwise expressly stated, such representative example and implementation is not intended to be limited to the specific example referenced or cited.

[0025] As used in this article, "between" is a range that includes the endpoints of the range. For example, a number between x and y explicitly includes the numbers x and y, as well as any numbers that fall within x and y.

[0026] As used herein, the term "adeno-associated virus (AAV)" refers to a small, replication-defective, non-enveloped virus containing single-stranded DNA, belonging to the family Parvoviridae and the genus Dependoparvovirus. To date, more than 10 AAV serotypes have been identified, with serotype AAV2 being the most thoroughly studied. 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 the capsid of a first serotype and the genome of a second serotype (e.g., pseudotype AAV2 / 5 would correspond to an AAV with the genome of serotype AAV2 and the capsid of AAV5).

[0027] As mentioned in this article, 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 numerous additional subtypes have been isolated from other mammals and birds. See, for example, Ishibashi et al., "Adenoviruses of animals," in *The Adenoviruses*, ed. Ginsberg, Plenum Press, New York, NY, pp. 497–562 (1984); Strauss, "Adenovirus infections in humans," in *The Adenoviruses*, ed. Ginsberg, Plenum Press, New York, NY, pp. 451–596 (1984). 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 exhibit different immunological properties and are therefore classified into various serotypes. Two human adenovirus serotypes, AV2 and AV5, have been extensively studied and provide much of the general information about adenoviruses.

[0028] Adeno-associated virus (AAV) has become the preferred vector for gene therapy in over 120 clinical trials worldwide. The rapidly growing demand for recombinant AAV necessitates an efficient and robust manufacturing platform. However, current methods for AAV production, including transient transfection and helper virus systems, are extremely costly and labor-intensive. Therefore, this article describes isolated nucleic acid molecules and plasmid- / helper virus-free AAV production cell lines and their usage methods for producing adeno-associated virus (AAV). This technological approach enables efficient AAV production, provides a long-term solution, and significantly reduces costs. The AAV production cell lines described in this article represent a next-generation platform for both clinical and commercial AAV manufacturing.

[0029] As used herein, “vector” or “expression vector” refers to a nucleic acid replicon, such as a plasmid, bacteriophage, virus, or granule, to which another nucleic acid segment may be attached to enable the attached nucleic acid segment to replicate and / or be expressed in a host cell. The term “vector” includes both attached (e.g., plasmid) and non-attached vectors. The term “vector” may also include synthetic vectors. Non-limiting examples of vectors include baculovirus vectors, bacteriophage vectors, plasmids, phage particles, granules, Fors plasmids, 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 specific vectors for a particular target host (e.g., mammalian cells, such as E1 complementary production cells described herein, including but not limited to HEK293 cells, PER.C6 cells, CAP® cells, and derivatives thereof). Vectors can be introduced into desired host cells (e.g., HEK293 cells, PER.C6 cells, CAP® cells, or derivatives thereof) using well-known methods (including but not limited to transfection, transduction, cell fusion, and lipid transfection). Vectors may contain various regulatory elements, including, for example, constitutive and inducible promoters, transcription enhancers, and transcription terminators.

[0030] As used herein, "isolated nucleic acid molecules" includes vectors and plasmids that may contain isolated nucleic acid molecules, as well as similar structures in which the isolated nucleic acid molecules can be manipulated, stored, transported, and ultimately used in various cell transfection systems. The isolated nucleic acid molecules described herein can be used to produce AAV as described herein, but also in various non-AAV producing cell lines (including transient transfection systems). The isolated nucleic acid molecules described herein suitably also include various additional elements and sequences required for use in the cell systems described herein (including mammalian cells).

[0031] "Nucleic acid," "nucleic acid molecule," or "oligonucleotide" refers to a polymer containing covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (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, miRNA, or MIRNA.

[0032] A "gene" refers to an assembly of nucleotides encoding a polypeptide and includes cDNA and genomic DNA nucleic acid molecules. A "gene" also refers to a nucleic acid fragment that can act as a regulatory sequence located before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. In some embodiments, a gene is integrated with multiple copies. In some embodiments, a gene is integrated at a predefined copy number.

[0033] As mentioned herein, the term "heterogeneous gene" or "HG," when referring to nucleic acid sequences (such as coding or control sequences), indicates nucleic acid sequences that are not typically linked together and / or not typically associated with a particular cell, such as genes. In some embodiments, a heterogeneous gene is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from those of a natural gene). As used herein, allelic variations or naturally occurring mutations do not produce heterogeneous DNA.

[0034] In the various embodiments described herein, nucleic acid molecules are capable of encoding a variety of genes. That is, nucleic acid molecules produce mRNA of the genes described herein during transcription, which is then translated into the desired or required protein.

[0035] The various nucleic acid molecules encoding the many genes described herein are suitably controlled by promoters. As used herein, “controlled by” means that a gene is regulated by a “promoter,” a “promoter sequence,” or a “promoter region,” which means that the DNA regulatory region / sequence can bind to RNA polymerase and initiate transcription of downstream coding or non-coding gene sequences. In other words, the promoter is operatively combined with or operatively linked to the gene. As mentioned herein, the terms “operatively combined,” “in an operationally possible order,” “operatively linked,” and “operatively” mean that the nucleic acid sequences are linked in such a way that they produce a promoter capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule.

[0036] In some instances of this disclosure, the promoter sequence includes a transcription start site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above background. In some embodiments, the promoter sequence includes a transcription start site and a protein-binding domain responsible for RNA polymerase binding. Eukaryotic promoters will often, but not always, contain both a "TATA" box and a "CAT" box. A variety of promoters, including inducible promoters, can be used to drive gene expression, for example, in the host cells or vectors of this disclosure. In some embodiments, the promoter is not a leaky promoter, i.e., the promoter does not constitutively express any of the gene products described herein. In other embodiments described herein, the promoter is a constitutive promoter that initiates mRNA synthesis without being influenced by external regulation.

[0037] Suitablely, the promoters used to control the transcription of the various genes required for AAV production as described herein are derepressed promoters. As used herein, a “derepressed promoter” is a structure comprising an inducible or functional promoter and an additional element or sequence capable of binding a repressive element to cause repression of that inducible or functional promoter. “Repression” refers to the reduction or inhibition of transcription initiation of a downstream coding or non-coding gene sequence by a promoter. A “repressive element” is a protein or polypeptide capable of binding to a promoter (or vicinity of a promoter) to reduce or inhibit promoter activity. A repressive element may interact with its substrate or binding partner, causing the repressive element to undergo a conformational change. This conformational change causes the repressive element to lose its ability to reduce or inhibit the promoter, resulting in “derepressed” promoter activity, thereby allowing the promoter to continue transcription initiation.

[0038] In some embodiments, a derepressed promoter comprises an inducible or functional promoter and one or more tetracycline operon sequences (TetO2). When the “inducible promoter” is affected by or exposed to a corresponding regulatory protein or other external stimulus (such as a chemical agent, stress, or biological stimulus), the promoter regulates (e.g., activates or inactivates) the transcriptional activity of the nucleic acid operatively linked to it. A “functional promoter” is a promoter that would enable transcription initiation in the absence of a repressive element. Various inducible and functional promoters that can be used to practice this invention are known in the art and include, for example, promoters of PCMV, PH1, P19, P5, P40, and adenovirus helper genes (e.g., E1A, E1B, E2A, E4Orf6, and VA).

[0039] As used herein, the terms “Tet operon sequence”, “Tet operon sequence motif”, “Tet operon”, “TetO2”, or “TetO” are intended to encompass all categories of Tet operon sequences, such as TetO(A), TetO(B), TetO(C), TetO(D), TetO(E), TetO(G), TetO(H), TetO(J), and TetO(Z). The nucleotide sequences of the Tet repressors and their corresponding Tet operon sequences of members of classes A, B, C, D, E, G, H, J, and Z are well known in the art, see, for example, Waters 1983, Null. Acids Res 11:6089-6105; Hillen 1983, Nucl. Acids Res. 11:525-539; Postle 1984, Nucl. Acids Res. 12:4849-4863; Unger 1984, Gene 31: 103-108; Unger 1984, Nucl Acids Res. 12:7693-7703; and Tovar 1988, Mol. Gen. Genet. 215:76-80, all of which are incorporated herein by reference in their entirety regarding the specific Tet operon sequences disclosed herein. The Tet operon sequence is also disclosed in U.S. Patent No. 5,464,758.

[0040] Exemplary repressor elements that can be used as derepressor promoters and their corresponding binding partners are known in the art, and include systems such as the cumate gene-switch system (CuO operon, CymR repressor and cumate binding partner) (see, for example, Mullick et al., “The cumate gene-switch: a system for regulated expression in mammalian cells,” BMC Biotechnology 6:43 (1-18) (2006), the disclosure of which is incorporated herein by reference in its entirety, including the disclosure of the derepressor promoter system described therein), and the TetO / TetR system described herein (see, for example, Yao et al., “Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells,” HumanGene Therapy 9:1939-1950 (1998), the disclosure of which is incorporated herein by reference in its entirety).

[0041] VA (virus-associated) RNA is a non-coding type found in adenoviruses. It plays a role in the regulation of translation. This RNA has two copies, called VAI or RNA I VA and VAII or RNA II VA. These two RNA VA genes are distinct genes in the adenovirus genome. RNA I VA is the dominant type, while RNA II VA is expressed at lower levels. Neither transcript is polyadenylated, and both are transcribed by PolIII.

[0042] As described herein, nucleic acid molecules suitably encode viral accessory genes. Viral accessory genes include various adenovirus genes, herpesvirus genes, and bocavirus genes (see, for example, Guido et al., “Human bocavirus: Current knowledge and future challenges,” World J. Gateroenterol 22:8684-8697, the disclosure of which is incorporated herein by reference in its entirety). In an exemplary embodiment, the viral accessory gene is an adenovirus accessory gene. As mentioned herein, the term “adenovirus accessory gene” or “AV accessory gene” refers to a gene consisting of one or more nucleic acid sequences derived from one or more adenovirus subtypes or serotypes that facilitates the replication and packaging of adeno-associated viruses. In some embodiments, the adenovirus accessory gene is E1A, E1B, E2A, E4 (including E4Orf6), VA, or a combination thereof, or any other adenovirus accessory gene. In an exemplary embodiment, the adenovirus accessory gene includes both the E2A gene and the E4Orf6 gene.

[0043] In other alternative embodiments, this document provides an isolated nucleic acid molecule operatively comprising: a first inducible promoter and two tetracycline operon sequences (TetO2 sequences); an E2A gene controlled by the first inducible promoter; a virus-associated (VA) noncoding RNA controlled by a second inducible promoter; a third inducible promoter and the TetO2 sequence; an E4 gene controlled by the third inducible promoter; and an antibiotic resistance gene.

[0044] In other embodiments, this disclosure provides an isolated nucleic acid molecule operatively comprising: a first inducible promoter and a TetO2 sequence; an E2A gene controlled by the first inducible promoter; a virus-associated (VA) non-coding RNA controlled by a second inducible promoter; a third inducible promoter and a TetO2 sequence; an E4 gene controlled by the third inducible promoter; an expression cassette of a protein ortholog and peptide tag encoding VA RNA controlled by a repressive promoter comprising a human cytomegalovirus (CMV) promoter and a TetO2 sequence; a termination sequence; and an antibiotic resistance gene.

[0045] As described herein, in exemplary embodiments, the E2A gene encoded by a nucleic acid molecule is suitably controlled by a naturally occurring first inducible promoter of the E2A gene. In some embodiments, the VA non-coding RNA encoded by a nucleic acid molecule is suitably controlled by a naturally occurring second inducible promoter of the VA non-coding RNA. In some embodiments, the second inducible promoter is the H1 promoter. In some embodiments, the E4 gene encoded by a nucleic acid molecule is suitably controlled by a naturally occurring third inducible promoter of the E4 gene.

[0046] As used herein, a protein ortholog is a protein that has the same specificity in different organisms, for example, by binding to the same ligands and similar DNA sites in the relevant genome. Therefore, orthologous proteins carry the same or similar specificity-determining residues. In some embodiments, the isolated nucleic acid molecules described herein comprise protein orthologs of VA RNA. In some embodiments, the protein orthologs are influenza nonstructural protein 1 (NS1), Ebola virus protein 35 (VP35), orthoreovirus σ3 protein (σ3), group C rotavirus nonstructural protein 3 (NSP3), vaccinia virus E3L protein (E3L), herpes simplex virus type 1 US11 protein (US11), Epstein-Barr virus SM protein (SM), baculovirus PK2 protein (PK2), hepatitis C virus nonstructural protein 5A protein (NS5A), human herpesvirus-8 protein vIRF-2 (vIRF-2), human immunodeficiency virus protein Tat, vaccinia virus K3L protein (K3L), herpes simplex virus protein γ134.5 / ICP34.5, or human papillomavirus-18 E6 protein (E6). In some embodiments, the protein ortholog is VP35. In some embodiments, the protein ortholog is SM.

[0047] Alternatively or additionally, the protein ortholog of the VA RNA of any isolated nucleic acid molecule of the present invention can be operatively linked to a peptide tag to assist in its isolation and detection, or to assist in the recognition and expression of the encoded protein. Various peptide tags can be used in the context of the present invention, including PK tags, FLAG tags, MYC tags, hemagglutinin (HA) tags, and multihistidine tags. Peptide tags can be diagnosed by immunoassay using anti-tag antibodies. In some embodiments, the peptide tag is located solely at the N-terminus of the protein ortholog of the VA RNA. In some embodiments, the isolated nucleic acid molecule contains a peptide protein tag. In some embodiments, the peptide protein tag is an HA tag. In some embodiments, the peptide protein tag is a FLAG tag. In some embodiments, the FLAG tag is an N-terminal FLAG tag.

[0048] As used herein, the role of a termination sequence is to define the ends of transcription units (such as genes) and initiate the process of releasing newly synthesized RNA from the transcription machinery. Terminators are found downstream of the gene to be transcribed and typically appear directly after any 3' regulatory element, such as polyadenylation or poly(A) signaling. In some embodiments, any of the nucleic acid molecules disclosed herein contains a termination sequence. In some embodiments, the termination sequence is bovine growth hormone polyadenylation (bgh-PolyA) signaling.

[0049] As mentioned herein, a "reporter gene" is a gene whose expression confers a phenotype that can be easily identified and measured in cells. In some embodiments, a reporter gene includes a fluorescent protein gene. In some embodiments, a reporter gene includes a selection gene.

[0050] In some embodiments, the isolated nucleic acid molecule contains a selection gene. As mentioned herein, the term "selection gene" refers to a gene encoding an enzyme activity that confers the ability to grow in a medium lacking nutrients that would otherwise be essential; additionally, a selection gene can confer resistance to antibiotics or drugs in cells expressing the selection gene. A selection gene can be used to confer a specific phenotype on host cells. When host cells must express the selection gene to grow in a selective medium, the gene is called a positive selection gene. A selection gene can also be used to select host cells containing a specific gene; a selection gene used in this manner is called a negative selection gene. In some embodiments, the selection gene is an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is a puromycin resistance gene. In some embodiments, the antibiotic resistance gene is an isoprothiolane resistance gene. In some embodiments, the antibiotic resistance gene is a hygromycin resistance gene. In some embodiments, the antibiotic resistance gene is a zeocin resistance gene.

[0051] In an exemplary embodiment, the nucleic acid molecule includes two inverted terminal repeat (ITR) sequences. As is known in the art, these ITR sequences (i.e., AAV2 ITRs) are single-stranded sequences of nucleotides, downstream of which is their inverse complement. The ITR sequences represent the minimum sequence required for the AAV genome to replicate, rescue, package, and integrate. Suitably, these ITR sequences are located flanking a target gene. Therefore, in embodiments, the nucleic acid molecule also encodes a target gene. This target gene may be, for example, a reporter gene, a selection gene, or a therapeutic target gene. In some embodiments, the nucleic acid sequence has transposon-specific inverted terminal repeat (ITR) sequences flanking both the 5' and 3' ends.

[0052] As used herein, a “core insulator” refers to a gene boundary element that blocks the interaction between an enhancer and a promoter. The presence of a core insulator between an enhancer and a promoter allows the insulator to block subsequent interactions. The core insulator determines which set of genes the enhancer can affect. Insulators are needed when two adjacent genes on a chromosome have very different transcriptional patterns and a mechanism needs to be induced or repressed without repressing the adjacent genes. In some embodiments, any of the nucleic acid sequences disclosed herein contains a core insulator sequence inserted downstream of the 3' ITR and upstream of the 5' ITR. In some embodiments, the nucleic acid molecule contains a core insulator sequence inserted between a termination sequence and an antibiotic resistance gene.

[0053] As used herein, the term "Rep gene" refers to a region of the AAV genome recognized in the art as encoding viral replication proteins that collectively participate in viral genome replication; or its functional homologs, such as the human herpesvirus 6 (HHV-6) rep gene, which is also known to mediate AAV2 DNA replication. Therefore, the Rep coding region may include genes encoding AAV Rep78 and Rep68 ("long forms of Rep") and Rep52 and Rep40 ("short forms of Rep"), or their functional homologs. As used herein, the Rep coding region may be derived from any viral serotype, such as the AAV serotype described herein. This region need not include all wild-type genes, but may be altered (e.g., through nucleotide insertion, deletion, or substitution) as long as the existing Rep genes provide sufficient integration function when expressed in suitable target cells. See, for example, Muzyczka, N., Current Topics in Microbiol. and Immunol. 158:97-129 (1992); and Kotin, RM, Human Gene Therapy 5:793-801 (1994).

[0054] In other alternative embodiments, this document provides an isolated nucleic acid molecule comprising a plasmid encoding the following elements: a first inducible promoter and a TetO2 sequence; a Rep78 gene containing a silenced p19 promoter located within the coding region of the Rep78 gene, wherein the silenced p19 promoter contains a mutation at the SP1, TATA-1, and / or TATA-2 sites; a second inducible promoter and a TetO2 sequence; a Rep52 gene controlled by the second inducible promoter; a termination sequence; and an antibiotic resistance gene.

[0055] In the context of the nucleic acid sequences described herein, the term "sequence identity" or "identity %" refers to the percentage of identical residues in the compared sequences when they are aligned within a specified comparison window. The comparison window can be a segment of at least 10 to over 1000 residues within which sequences can be aligned and compared. Alignment methods used to determine sequence identity are well-known and can be performed using publicly available databases such as BLAST (blast.ncbi.nlm.nih.gov / Blast.CGI.).

[0056] The Kozak concordant sequence, Kozak concordant region, or Kozak sequence is a sequence known to be present on eukaryotic mRNA and has a concordant sequence (gcc) gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". In some embodiments, the isolated nucleic acid molecules disclosed herein contain the Kozak concordant sequence. In some embodiments, the nucleic acid molecules contain a concordant sequence having at least about 70%, at least about 80%, at least about 90% sequence identity or higher with the Kozak concordant sequence. In some embodiments, the isolated nucleic acid molecules contain the Kozak concordant sequence after a gene cassette encoding one or more target proteins is inserted into a vector (e.g., inserted at a restriction site downstream of a promoter). For example, the isolated nucleic acid molecule may contain the nucleotide sequence GCCGCCATG, where ATG is the start codon of the target protein. In some embodiments, the isolated nucleic acid molecule contains the nucleotide sequence GCGGCCGCCATG, where ATG is the start codon of the target protein. In some embodiments, the Rep78 gene of the isolated nucleic acid molecule also contains a Kozak co-occurring sequence. In some embodiments, the Rep52 gene of the isolated nucleic acid molecule also contains a Kozak co-occurring sequence.

[0057] To more effectively control the production of the Rep protein, non-classical start codons or non-classical translation start codons can be used. In some embodiments, the Rep78 gene of the isolated nucleic acid molecule also contains a non-classical start codon. In some embodiments, the Rep52 gene of the isolated nucleic acid molecule also contains a non-classical start codon. In some embodiments, the non-classical translation start codon is GTG, CTG, ACG, or TTG. In some embodiments, the non-classical translation start codon is CTG.

[0058] Non-limiting examples of gene modifications that can be used in this invention include codon optimization aimed at, for example, modifying non-CTG leucine codons to CTG, or non-AAG lysine codons to AAG. Another example of nucleic acid codon optimization modification is increasing GC content. To improve vector stability and Rep52 expression, the DNA sequence of the Rep52 coding region is optimized for human cell codon usage while retaining the same protein sequence, which significantly reduces the DNA sequence identity between the Rep52 coding sequence and the Rep78 coding sequence from 100% sequence identity to 80.1% sequence identity. In some embodiments, the Rep52 of any of the nucleic acid molecules disclosed herein is codon-optimized. In some embodiments, the VP1 gene of any of the nucleic acid molecules disclosed herein is optimized, excluding the first 439 bp of the coding region.

[0059] In other embodiments, the first inducible promoter of the nucleic acid molecule disclosed herein is a human cytomegalovirus (CMV) immediate early enhancer-promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter. In some embodiments, the first inducible promoter is an RSV promoter. In some embodiments, the first inducible promoter is a UbC promoter. As described herein, the use of artificial introns allows for the removal of the inducible promoter after inducible promoter and before AAV production. In some embodiments, the nucleic acid molecule also includes an intron of the UbC promoter inserted upstream of the UbC promoter.

[0060] In some embodiments, the second inducible promoter of the nucleic acid molecule disclosed herein is the human CMV immediate early enhancer-promoter, the chicken β-actin promoter (CAG) fused with the CMV immediate early enhancer, the human ubiquitin C (UbC) promoter, or the Rous sarcoma virus (RSV) promoter. In some embodiments, the second inducible promoter is the CAG promoter. In some embodiments, the second inducible promoter is the CMV promoter.

[0061] As mentioned herein, the term "Cap gene" refers to a region in the AAV genome that is recognized in the art as encoding a viral capsid protein. Illustrative (non-limiting) examples of such capsid proteins are AAV capsid proteins VP1, VP2, and VP3. The Cap gene used in this disclosure may be derived from any AAV serotype or combination of AAV serotypes.

[0062] In other embodiments, this disclosure provides an isolated nucleic acid molecule operably comprising: a first inducible promoter and a TetO2 sequence; a VP1 gene; a second inducible promoter and a TetO2 sequence; VP2 and VP3 genes; and an antibiotic resistance gene. In some embodiments, the nucleic acid sequence includes a core insulator sequence inserted between 1) the VP1 gene and 2) the second inducible promoter and the TetO2 sequence. In some embodiments, the nucleic acid sequence includes a core insulator sequence inserted between 1) the VP2 and VP3 genes and 2) the antibiotic resistance gene.

[0063] As mentioned herein, the terms "target gene," "therapeutic target gene," or "GOI" are used to describe heterologous genes. GOIs disclosed herein may include any desired gene encoding a defective or missing protein in the genome of a target cell, or a non-natural protein encoding a desired biological or therapeutic effect (e.g., antiviral function), or the sequence may correspond to a molecule with antisense or ribozyme function. Representative (non-limiting) examples of suitable therapeutic target genes include those used to treat inflammatory diseases, autoimmune diseases, chronic diseases, and infectious diseases, including: conditions such as AIDS, cancer, neurological disorders, cardiovascular diseases, and hypercholesterolemia; various blood disorders, including various anemias, thalassemia, and hemophilia; and genetic defects such as cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, etc. Several antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of mRNA) have been described in the art for antisense therapy of cancer and viral diseases and are also examples of suitable therapeutic target genes.

[0064] In another embodiment, the nucleic acid molecule contains a target gene (GOI) located downstream of the VP2 and VP3 genes and upstream of the antibiotic resistance gene. In some embodiments, the nucleic acid molecule contains a core insulator sequence inserted between the VP3 gene and the GOI. In some embodiments, the nucleic acid molecule contains a core insulator sequence inserted between the GOI and the antibiotic resistance gene. In some embodiments, the nucleic acid molecule contains core insulator sequences inserted between the VP3 gene and the GOI, and between the GOI and the antibiotic resistance gene. In some embodiments, the GOI is a therapeutic gene.

[0065] In some embodiments, this disclosure provides a mammalian cell for producing adeno-associated virus (AAV) comprising any of the isolated nucleic acid molecules disclosed herein integrated into its genome. In some embodiments, the mammalian cell further comprises a nucleic acid encoding a transcriptional repression domain co-located with a nucleic acid encoding a tetracycline repressor protein. In some embodiments, the mammalian cell disclosed herein further comprises a nucleic acid molecule encoding a target gene.

[0066] The AAV production cells described in this paper provide a long-term and cost-effective solution for large-scale AAV manufacturing. Because constitutive expression of accessory or Rep proteins can be cytotoxic, the strategy described in this paper allows for control of their expression via engineered derepressed promoters.

[0067] As used herein, the term "mammalian cell" includes cells from any member of the mammalian order, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. In some embodiments, the cells are mouse cells, human cells, Chinese hamster ovary (CHO) cells, CHOK1 cells, CHO-DXB11 cells, CHO-DG44 cells, CHOK1SV cells (including all variants (e.g., POTELLIGENT®, Lonza, Slough, UK)), and CHOK1SV GS-KO (glutamine synthase knockout) cells (including all variants (e.g., XCEED™ Lonza, Slough, UK)). In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the mammalian cell is a human cell.

[0068] Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK293, HeLa cells, or HT1080 cells. As used herein, the term "human embryonic kidney 293 (HEK293) cell" refers to a cell line originally derived from human embryonic kidney cells and containing approximately 4.5 kb of the Ad5 genome. Variants of HEK293 cells have been developed and include, for example, HEK293S, HEK293T, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEK293MSR, and HEK293A. As used herein, the term "HEK293 cell" encompasses all HEK293 cell variants and derivatives, including but not limited to the variants described herein. See, for example, Yuan et al., “The Scattered Twelve Tribes of HEK293,” Biomed Pharmacol J 2018; 11(2). In some embodiments, the human cells are HEK cells. In some embodiments, the HEK cells are HEK293 cells.

[0069] As described herein, mammalian cells are suitable mammalian cell cultures, and in embodiments, may be suspension cultures. As described herein, the use of suspension cell cultures allows for increased scalability and yield of AAVs. Adherent cultures refer to cells that grow on a substrate surface (e.g., plastic plates, culture dishes, or other suitable cell culture growth platforms) and may exhibit anchorage dependence. Suspension cultures refer to cells that can be maintained in, for example, culture flasks or large suspension culture vessels, which allows for a large surface area for gas and nutrient exchange. Suspension cell cultures typically utilize stirring or agitation mechanisms to provide adequate mixing. Culture media and conditions used to maintain cell suspension are well known in the art. An exemplary suspension cell culture comprises human HEK293 clone cells.

[0070] As used herein, “transfection” means the introduction of a foreign nucleic acid molecule (including a vector) into a cell. A “transfected” cell contains a foreign nucleic acid molecule inside the cell, and a “transformed” cell is a cell in which the foreign nucleic acid molecule induces a change in cell phenotype. Transfected nucleic acid molecules can be integrated into the host cell’s genomic DNA and / or can be maintained by the cell outside the chromosome, temporarily or for a long time. A host cell or organism that expresses a foreign nucleic acid molecule or fragment is referred to as a “recombinant” organism, a “transformed” organism, or a “transgenic” organism. Various transfection techniques are well known in the art. See, for example, 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). Such technologies can be used to introduce one or more exogenous DNA motifs, such as AAV vector cassettes, AAV helper constructs, and other nucleic acid molecules, into suitable host cells.

[0071] Various methods for transfecting mammalian cells with the isolated nucleic acid molecules (i.e., vectors) described herein are 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 delivery systems, etc.

[0072] In other additional embodiments, this disclosure provides a method for producing adeno-associated virus (AAV), the method comprising inducing the production of a genome-integrated nucleic acid sequence of any of the mammalian cells disclosed herein, culturing the mammalian cells, and harvesting the AAV.

[0073] As discussed herein, the production method and mammalian cells of this disclosure advantageously provide high titers of AAV produced by mammalian cells. In some embodiments, the amount of AAV produced is about 10... 10 To about 10 11 One viral genome / mL (vg / mL). In some implementations, the amount of AAV produced is at least about 10 10 One viral genome / mL (vg / mL). In some implementations, the amount of AAV produced is at least about 10 11 Viral genomes / mL (vg / mL).

[0074] In some embodiments, the mammalian cells provided herein are substantially free of helper viruses. As mentioned herein, a "helper virus" is any non-AAV virus added to enable adeno-associated virus (AAV) replication and packaging. Representative (non-limiting) examples of helper viruses are adenoviruses and herpesviruses. In some embodiments, the term "substantially free of helper viruses" means cells having fewer than 100, fewer than 10, or fewer than 1 helper virus per cell. In some embodiments, the term "substantially free of helper viruses" means cells or cell populations where helper viruses are absent using detection methods known to those skilled in the art. In some embodiments, the cells are free of wild-type helper viruses. In some embodiments, the term "wild-type virus" means any intact non-AAV virus capable of replicating independently of any other virus in a cell.

[0075] The method for producing AAV can be used in a continuous manufacturing system. In an exemplary embodiment, the use of suspension cell cultures allows for the production of large quantities of AAV with high productivity and extended culture conditions, thereby allowing for multiple harvests of AAV from each batch of starting cells.

[0076] The production method may utilize any suitable reactor, including but not limited to stirred tanks, airlift reactors, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or sputtered bed bioreactors. As used herein, "reactor" may include fermenters or fermentation units or any other reaction vessel, and the terms "reactor" and "fermenter" are used interchangeably. The terms "fermenter" or "fermentation" refer to both microbial cultures and mammalian cultures. For example, in some aspects, an example bioreactor unit may perform one or more or all of the following: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inflow and outflow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Example reactor units, such as fermentation units, may contain multiple reactors within a unit. For example, 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 in each unit, and / or the facility may contain multiple units having one or more reactors within the facility. In various embodiments, the bioreactors may be suitable for batch, semi-feed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the volume of the bioreactor may be between about 100 mL and about 50,000 L. Non-limiting examples of volume are 100 mL, 250 mL, 500 mL, 750 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 15 L, 20 L, 25 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, 500 L, and 550 L. Capacities range from 100 liters to 500 liters. Suitable reactors can be multiple-use, single-use, disposable, or non-disposable, and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and / or glass.

[0077] Other exemplary implementations

[0078] Implementation scheme 1 is an isolated nucleic acid molecule operatively comprising: a first inducible promoter and two tetracycline operon sequences (TetO2 sequences); an E2A gene controlled by the first inducible promoter; a virus-associated (VA) noncoding RNA controlled by a second inducible promoter; a third inducible promoter and a TetO2 sequence; an E4 gene controlled by the third inducible promoter; and an antibiotic resistance gene.

[0079] Implementation scheme 2 includes the nucleic acid molecule as described in implementation scheme 1, wherein the first inducible promoter is natural for the E2A gene.

[0080] Implementation scheme 3 includes a nucleic acid molecule as described in implementation scheme 1 or 2, wherein the second inducible promoter is a natural promoter of the VA noncoding RNA.

[0081] Implementation scheme 4 includes a nucleic acid molecule as described in any one of implementation schemes 1 to 3, wherein the second inducible promoter is an H1 promoter.

[0082] Implementation scheme 5 includes a nucleic acid molecule as described in any one of implementation schemes 1 to 4, wherein the third inducible promoter is natural for the E4 gene.

[0083] Implementation scheme 6 includes a nucleic acid molecule as described in any one of implementation schemes 1 to 5, wherein the antibiotic resistance gene is a puromycin resistance gene.

[0084] Implementation scheme 7 includes a nucleic acid molecule as described in any one of implementation schemes 1 to 6, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the E4 gene and the antibiotic resistance gene.

[0085] Implementation scheme 8 includes a nucleic acid molecule as described in any one of implementation schemes 1 to 7, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeat (ITR) sequences at both the 5' and 3' ends.

[0086] Implementation scheme 9 includes a nucleic acid molecule as described in implementation scheme 8, wherein the nucleic acid sequence is contained in a core insulator sequence inserted downstream of the 3' end of the ITR and upstream of the 5' end of the ITR.

[0087] Implementation scheme 10 includes a nucleic acid molecule as described in any one of implementation schemes 1 to 9, which further comprises multiple copies of VA noncoding RNA.

[0088] Implementation scheme 11 is an isolated nucleic acid molecule operatively comprising: a first inducible promoter and a TetO2 sequence; an E2A gene controlled by the first inducible promoter; a virus-associated (VA) non-coding RNA controlled by a second inducible promoter; a third inducible promoter and a TetO2 sequence; an E4 gene controlled by the third inducible promoter; an expression cassette of a protein ortholog and a peptide tag encoding the VA RNA controlled by a repressor promoter, the repressor promoter comprising a human cytomegalovirus (CMV) promoter and a TetO2 sequence; a termination sequence; and an antibiotic resistance gene.

[0089] Implementation scheme 12 includes a nucleic acid molecule as described in implementation scheme 11, wherein the first inducible promoter is natural for the E2A gene.

[0090] Implementation scheme 13 includes a nucleic acid molecule as described in implementation scheme 11 or 12, wherein the second inducible promoter is a natural promoter of the VA noncoding RNA.

[0091] Implementation scheme 14 includes a nucleic acid molecule as described in any one of implementation schemes 11 to 13, wherein the second inducible promoter is an H1 promoter.

[0092] Implementation scheme 15 includes an isolated nucleic acid molecule as described in any one of implementation schemes 12 to 14, wherein the third inducible promoter is natural for the E4 gene.

[0093] Implementation scheme 16 includes isolated nucleic acid molecules as described in any one of implementation schemes 11 to 15, wherein the orthologs of the proteins are influenza nonstructural protein 1 (NS1), Ebola virus protein 35 (VP35), orthoreovirus σ3 protein (σ3), group C rotavirus nonstructural protein 3 (NSP3), vaccinia virus E3L protein (E3L), herpes simplex virus type 1 US11 protein (US11), Epstein-Barr virus SM protein (SM), baculovirus PK2 protein (PK2), hepatitis C virus nonstructural protein 5A protein (NS5A), human herpesvirus-8 protein vIRF-2 (vIRF-2), human immunodeficiency virus protein Tat, vaccinia virus K3L protein (K3L), herpes simplex virus protein γ134.5 / ICP34.5, and human papillomavirus-18 E6 protein (E6).

[0094] Implementation scheme 17 includes isolated nucleic acid molecules as described in implementation scheme 16, wherein the protein ortholog is VP35.

[0095] Implementation scheme 18 includes isolated nucleic acid molecules as described in implementation scheme 16, wherein the protein ortholog is SM.

[0096] Implementation scheme 19 includes isolated nucleic acid molecules as described in any one of implementation schemes 11 to 18, wherein the peptide protein tag is a FLAG tag.

[0097] Implementation scheme 20 includes isolated nucleic acid molecules as described in implementation scheme 19, wherein the FLAG tag is an N-terminal Flag tag.

[0098] Implementation scheme 21 includes an isolated nucleic acid molecule as described in any one of implementation schemes 11 to 20, wherein the termination sequence is a bovine growth hormone polyadenylation (bgh-PolyA) signal.

[0099] Implementation scheme 22 includes a nucleic acid molecule as described in any one of implementation schemes 11 to 21, wherein the antibiotic resistance gene is a puromycin resistance gene.

[0100] Implementation scheme 23 includes a nucleic acid molecule as described in any one of implementation schemes 11 to 22, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the E4 gene and the expression cassette.

[0101] Implementation scheme 24 includes a nucleic acid molecule as described in any one of implementation schemes 11 to 23, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeat (ITR) sequences at both the 5' and 3' ends.

[0102] Implementation scheme 25 includes a nucleic acid molecule as described in implementation scheme 24, wherein the nucleic acid sequence is contained in a core insulator sequence inserted downstream of the 3' end of the ITR and upstream of the 5' end of the ITR.

[0103] Implementation scheme 26 is an isolated nucleic acid molecule operatively comprising a plasmid encoding the following elements: a first inducible promoter and a TetO2 sequence; a Rep78 gene containing a silenced p19 promoter located within the coding region of the Rep78 gene, wherein the silenced p19 promoter contains a mutation at the SP1, TATA-1, and / or TATA-2 sites; a second inducible promoter and a TetO2 sequence; a Rep52 gene controlled by the second inducible promoter; a termination sequence; and an antibiotic resistance gene.

[0104] Implementation scheme 27 includes isolated nucleic acid molecules as described in implementation scheme 26, wherein the Rep78 gene further contains a Kozak common sequence or a non-classical start codon.

[0105] Implementation scheme 28 includes a nucleic acid molecule as described in implementation scheme 26, wherein the Rep52 gene further contains a Kozak common sequence or a non-classical start codon.

[0106] Implementation scheme 29 includes an isolated nucleic acid molecule as described in any one of implementation schemes 26 to 28, wherein the first inducible promoter is a human cytomegalovirus (CMV) immediate early enhancer promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

[0107] Implementation scheme 30 includes isolated nucleic acid molecules as described in implementation scheme 29, wherein the first inducible promoter is the RSV promoter.

[0108] Implementation scheme 31 includes an isolated nucleic acid molecule as described in any one of implementation schemes 26 to 30, wherein the second inducible promoter is a human CMV immediate early enhancer-promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

[0109] Implementation scheme 32 includes isolated nucleic acid molecules as described in implementation scheme 31, wherein the second inducible promoter is a CAG promoter.

[0110] Implementation scheme 33 includes an isolated nucleic acid molecule as described in any one of implementation schemes 26 to 32, wherein the termination sequence is a bovine growth hormone polyadenylation (bgh-PolyA) signal.

[0111] Implementation scheme 34 includes a nucleic acid molecule as described in any one of implementation schemes 26 to 33, wherein the antibiotic resistance gene is a blast fungicide resistance gene.

[0112] Implementation scheme 35 includes a nucleic acid molecule as described in any one of implementation schemes 26 to 34, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the termination sequence and the antibiotic resistance gene.

[0113] Implementation scheme 36 includes a nucleic acid molecule as described in any one of implementation schemes 26 to 35, wherein the Rep52 is codon-optimized.

[0114] Implementation scheme 37 includes a nucleic acid molecule as described in any one of implementation schemes 26 to 36, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeat (ITR) sequences at both the 5' and 3' ends.

[0115] Implementation scheme 38 includes a nucleic acid molecule as described in implementation scheme 37, wherein the nucleic acid sequence comprises a core insulator sequence inserted downstream of the 3' end of the ITR and upstream of the 5' end of the ITR.

[0116] Implementation scheme 39 is an isolated nucleic acid molecule operatively comprising: a first inducible promoter and a TetO2 sequence; a VP1 gene; a second inducible promoter and a TetO2 sequence; VP2 and VP3 genes; and an antibiotic resistance gene.

[0117] Implementation scheme 40 includes isolated nucleic acid molecules as described in implementation scheme 39, wherein the VP1 gene is optimized, but excluding the first 439 bp of the coding region.

[0118] Implementation scheme 41 includes isolated nucleic acid molecules as described in implementation scheme 39 or 40, wherein the first inducible promoter is a human cytomegalovirus (CMV) immediate early enhancer promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

[0119] Implementation scheme 42 includes isolated nucleic acid molecules as described in implementation scheme 41, wherein the first inducible promoter is the UbC promoter.

[0120] Implementation scheme 43 includes the isolated nucleic acid molecule as described in implementation scheme 42, which further includes an intron of the UbC promoter inserted upstream of the UbC promoter.

[0121] Implementation scheme 44 includes an isolated nucleic acid molecule as described in any one of implementation schemes 37 to 43, wherein the second inducible promoter is a human CMV immediate early enhancer-promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

[0122] Implementation scheme 45 includes isolated nucleic acid molecules as described in implementation scheme 44, wherein the second inducible promoter is a CMV promoter.

[0123] Implementation scheme 46 includes an isolated nucleic acid molecule as described in any one of implementation schemes 39 to 45, which further includes a target gene (GOI) located downstream of the VP3 gene and upstream of the antibiotic resistance gene.

[0124] Implementation scheme 47 includes isolated nucleic acid molecules as described in implementation scheme 46, wherein the nucleic acid molecules contain core insulator sequences inserted between the VP3 gene and the GOI and between the GOI and the antibiotic resistance gene.

[0125] Implementation scheme 48 includes isolated nucleic acid molecules as described in implementation scheme 47, wherein the GOI is a therapeutic gene.

[0126] Implementation scheme 49 includes a nucleic acid molecule as described in any one of implementation schemes 39 to 48, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the VP1 gene and the second inducible promoter and the TetO2 sequence.

[0127] Implementation scheme 50 includes a nucleic acid molecule as described in any one of implementation schemes 39 to 49, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the VP2 and VP3 genes in d) and the antibiotic resistance gene in e).

[0128] Implementation scheme 51 includes a nucleic acid molecule as described in any one of implementation schemes 39 to 50, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeat (ITR) sequences at both the 5' and 3' ends.

[0129] Implementation scheme 52 includes a nucleic acid molecule as described in implementation scheme 51, wherein the nucleic acid sequence is contained in a core insulator sequence inserted downstream of the 3' end of the ITR and upstream of the 5' end of the ITR.

[0130] Implementation scheme 53 includes mammalian cells for producing adeno-associated virus (AAV) containing isolated nucleic acid molecules as described in any one of implementation schemes 1 to 52, which are integrated into their genome.

[0131] Implementation scheme 54 includes a mammalian cell as described in implementation scheme 53, which further includes a nucleic acid encoding a transcriptional repression domain in the same frame as the nucleic acid encoding a tetracycline repressor protein.

[0132] Embodiment 55 includes mammalian cells as described in Embodiment 53 or 54, wherein the mammalian cells are mammalian cell cultures.

[0133] Implementation scheme 56 includes mammalian cells as described in any one of implementation schemes 53 to 55, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

[0134] Embodiment 57 includes mammalian cells as described in any one of embodiments 53 to 55, wherein the mammalian cells are human cells.

[0135] Implementation scheme 58 includes mammalian cells as described in implementation scheme 57, wherein the human cells are human embryonic kidney (HEK) cells.

[0136] Embodiment 59 includes mammalian cells as described in Embodiment 58, wherein the HEK cells are HEK293 cells.

[0137] Implementation scheme 60 includes a mammalian cell as described in any one of implementation schemes 53 to 59, which further contains a nucleic acid molecule encoding the target gene.

[0138] Implementation Scheme 61 includes a method for producing adeno-associated virus (AAV), the method comprising inducing the production of a genome-integrated nucleic acid sequence in any of the mammalian cells described in Implementation Schemes 53 to 60, culturing the mammalian cells, and harvesting the AAV.

[0139] Implementation scheme 62 includes the method as described in implementation scheme 61, wherein the amount of AAV generated is approximately 10. 10 To about 10 11 Viral genomes / mL (vg / mL).

[0140] Implementation scheme 63 includes the method as described in implementation scheme 62, wherein the amount of AAV generated is at least about 10. 10 Viral genomes / mL (vg / mL).

[0141] Example

[0142] Example 1: Preparation of stable HEK293 clones expressing TetR-KRAB

[0143] One of the earliest Tet-On inducible systems utilized a tetracycline repressor (TetR) to silence the human cytomegalovirus (CMV) promoter by inserting two tetracycline operon sequences (TetO2) between the TATA box and the transcription start site (TSS) (Yao et al., “Tetracycline repressor, tetR, rather than the tetR-mammalian celltranscription factor fusion derivatives, regulates inducible gene expression in mammalian cells,” Hum Gene Ther. 9(13):1939-50 (1998)). In the absence of doxycycline, transcription initiation was blocked by the binding of TetR to the TetO2 site. When doxycycline was added to the culture medium, it competitively bound TetR and altered its conformation. This resulted in the release of TetR and the derepression / activation of the CMV promoter, leading to induced gene expression (see [link to article]). Figure 1 However, the high level of leakage expression of the induced protoxic protein in the original system poses a significant challenge to the stability of the generated inducible cell lines and limits their application in AAV production cell lines.

[0144] Improvements were made to the Tet-On inducible system to minimize leakage. First, as described by Suzlc et al., “A versatile tool for conditional gene expression and knockdown,” Nat Methods 3(2):109-16 (2006), an enhanced form of TetR was applied. In short, the strong repressive domain of KRAB was fused in-frame to the C-terminal portion of the original TetR, which significantly enhanced its repressive activity and minimized pre-induction basal gene expression. An SV40 nuclear localization signal (NLS) was also inserted to facilitate nuclear entry of the larger TetR-KRAB fusion protein (see [link to original text]). Figure 2A and Figure 2B Secondly, the expression cassette was optimized for high-level expression of the TetR-KRAB protein, incorporating a strong human CMV promoter to drive high levels of mRNA expression, a shared Kozak sequence to promote protein translation initiation, and a rabbit β-globin intron to increase mRNA levels. The cassette is further protected at both ends by two chicken hypersensitive site-4 (cHS4) extended core chromatin insulators, preventing undesirable silencing of the CMV promoter due to diffusion from neighboring heterochromatin. A Zeocin antibiotic resistance gene driven by the SV40 promoter was included for stable selection. Thirdly, using this optimized construct, a stable TetR-KRAB overexpressing clone HEK293 cell line, C20, was established via random plasmid integration. When compared with a commercially available stable TetR HEK293 cell line in transient transfection assays, the C20 cell line reduced AAV leakage yield to approximately 1 / 200.

[0145] sequence list

[0146] Any nucleic acid and amino acid sequences listed in this document or in the appended sequence listings are indicated using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 CFR § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is to be understood to be included by any reference to the strand shown.

[0147] The sequence of the pcDNA-TetR-Ins vector is provided below:

[0148] pcDNA-TetR-Ins (7147 bp) (SEQ ID NO: 1)

[0149]

[0150] The sequence of the pcDNA-TetR-KRAB-Ins vector is provided below:

[0151] pcDNA-TetR-KRAB-Ins (7493 bp) (SEQ ID NO: 2)

[0152]

[0153] Example 2: Design and validation of derepressed helper genes

[0154] pPBBG-iHelper2.0-HA vector

[0155] To drive the expression of adenovirus helper genes E2A and E4Orf6, two tetracycline operon sequences (TetO2) were inserted between the TATA box and the transcription start site (TSS) in the natural promoters of the E2A and E4 genes. VA RNA expression was driven by its natural promoter or by the H1 promoter with or without the TetO2 insertion. The VA gene promoter was embedded into the gene body of the non-coding RNA, and the TetO2 sequence was added upstream of the VA gene. To facilitate the detection of E4orf6 protein expression, the HA tag was fused to the N-terminal frame of the E4orf6 coding sequence. The expression cassettes of inducible E2A, E4, and VA as a single gene block were flanked by core insulator sequences, which are long-range regulatory sequences used to prevent the gene promoter from being activated by neighboring distal enhancers or from being silenced by heterochromatin diffusion, respectively. This sequence was fully synthesized and subcloned into the piggyBac transposon vector between the 5' and 3' transposon-specific ITRs. To allow for antibiotic selection of recombinant transposons in cells, a puromycin antibiotic resistance gene driven by the CMV promoter is also included, with a core insulator side-attached within the ITR. This produces the pPBBG-iHelper2.0-HA vector (see [link to vector]). Figure 3A ).

[0156] To enhance VA RNA expression in the pPBBG-iHelper2.0-HA vector design, a series of modifications were applied to the VA cassette in the pPBBG-iHelper2.0-HA vector design.

[0157] In the iHelper2.1 vector, both the TetO2 and VAII regions, originally designed in the pPBBG-iHelper2.0-HA vector, were removed (see [link to iHelper2.1 vector design]). Figure 3B In the iHelper2.2 vector, the VAI box from iHelper2.1 is repositioned between the E2A and E4 boxes to achieve inducibility from the neighboring TetO2 sequence (see [link]). Figure 3C In the iHelper2.3 vector, a constitutive H1 promoter is added upstream of the VAI box in iHelper2.1 to enhance VA expression (see [link to iHelper2.3 vector]). Figure 3D In the iHelper 2.4 vector, the VAI and H1 promoters from iHelper 2.3 are repositioned between the E2A and E4 boxes (see [link]). Figure 3ETo further enhance VA expression, the VA box was correspondingly increased from a single copy in iHelper 2.1, 2.2, 2.3, and 2.4 vectors to four tandem repeats in iHelper 2.5, 2.6, 2.7, and 2.8. The addition of additional promoters (H1) at different positions was also investigated. (See [link to related documentation]) Figure 3F – Figure 3I ).

[0158] To further enhance auxiliary functions, functional protein orthologs of VA RNA were screened to enhance AAV production. These protein-coding genes are derived from different viruses and are reported to function as inhibitors of protein kinase R (PKR), a key function of VA RNA (Langland et al., “Inhibition of PKR by RNA and DNA viruses,” Virus Res. 119(1):100-10 (2006); and Feng et al., “The VP35 protein of Ebola virus inhibits the antiviral effect mediated by double-stranded RNA-dependent protein kinase PKR,” J Virol. 81(1):182-92 (2007). VP35 from Ebola virus and SM from Epstein-Barr virus are two candidates that promote AAV production. To incorporate protein orthologs into the iHelper2.2 vector, the VP35 or SM gene was fused into the N-terminal Flag tag frame and placed under an inducible human CMV promoter with a TetO2 insertion, followed by BGH. Poly(A) signal. Then, the expression cassette was inserted into the region between E4 and the puromycin resistance gene in iHelper2.2, with a core insulator attached laterally. The iHelper3.1 vector containing the VP35 gene is shown in... Figure 4A In the middle, the iHelper3.2 vector containing the SM gene is shown in Figure 4B middle.

[0159] sequence list

[0160] The sequence of the pPBBG-iHelper2.0-HA vector is provided below:

[0161] pPBBG-iHelper2.0-HA (16,549 bp) (SEQ ID NO: 3)

[0162]

[0163] The sequence of the pPBBG-iHelper2.1-HA vector is provided below:

[0164] pPBBG-iHelper2.1-HA (15,878 bp) (SEQ ID NO: 4)

[0165]

[0166] The sequence of the pPBBG-iHelper2.2-HA vector is provided below:

[0167] pPBBG-iHelper2.2-HA (15,878 bp) (SEQ ID NO: 5)

[0168]

[0169] The sequence of the pPBBG-iHelper2.3-HA vector is provided below:

[0170] pPBBG-iHelper2.3-HA (15,992 bp) (SEQ ID NO: 6)

[0171]

[0172] The sequence of the pPBBG-iHelper2.4-HA vector is provided below:

[0173] pPBBG-iHelper2.4-HA (15, 992 bp) (SEQ ID NO: 7)

[0174]

[0175] The sequence of the pPBBG-iHelper2.5-HA vector is provided below:

[0176] pPBBG-iHelper2.5-HA (16, 658 bp) (SEQ ID NO: 8)

[0177]

[0178] The sequence of the pPBBG-iHelper2.6-HA vector is provided below:

[0179] pPBBG-iHelper2.6-HA (16,658 bp) (SEQ ID NO: 9)

[0180]

[0181] The sequence of the pPBBG-iHelper2.7-HA vector is provided below:

[0182] pPBBG-iHelper2.7-HA (17,114 bp) (SEQ ID NO: 10)

[0183]

[0184] The sequence of the pPBBG-iHelper2.8-HA vector is provided below:

[0185] pPBBG-iHelper2.8-HA (17,114 bp) (SEQ ID NO: 11)

[0186]

[0187] The sequence of the pPBBG-iHelper3.1-HA vector is provided below:

[0188] pPBBG-iHelper3.1-HA (18, 311 bp) (SEQ ID NO: 12)

[0189]

[0190] The sequence of the pPBBG-iHelper3.2-HA vector is provided below:

[0191] pPBBG-iHelper3.2-HA (18, 728 bp) (SEQ ID NO: 13)

[0192]

[0193] Example 3: Design and validation of the inducible Rep gene

[0194] Producing inducible Rep proteins can be challenging due to the critical maintenance of the Rep78 to Rep52 protein expression ratio required for high-titer AAV quality and quantity. Furthermore, the p19 promoter required for Rep52 expression is embedded in the coding region of Rep78, making direct promoter engineering difficult. To overcome these challenges, two strategies were developed.

[0195] First, the expression cassettes of Rep78 and Rep52 were separated from the overlapping genes into two separate expression cassettes, each driven by one of the following viral or non-viral promoters with varying strengths: human cytomegalovirus (CMV) immediate early enhancer-promoter, CMV immediate early enhancer fusion with chicken β-actin promoter (CAG), human ubiquitin C (UbC) promoter, and Rous sarcoma virus (RSV) promoter. To convert these constitutive promoters into inducible forms, two tetracycline operon sequences (TetO2) were inserted between the TATA cassette and the transcription start site (TSS).

[0196] By altering six nucleotides in three core regulatory elements (SP1, TATA-1, and TATA-2 sites) required for p19 activity, the original p19 promoter in the Rep78 open reading frame (ORF) was silenced. These changes did not alter the Rep78 protein sequence. To improve vector stability and Rep52 expression, the DNA sequence of the Rep52 coding region was optimized for human cell codon usage while preserving the same protein sequence, significantly reducing the sequence identity between Rep52 and Rep78 from 100% to 80.1%.

[0197] To improve Rep gene expression, Kozak sequences or the non-canonical start codon ACG were used to enhance or degrade protein expression, respectively. Sixteen inducible Rep vectors were designed in the piggyBac transposon vector. These vectors have a core insulator with Rep78 and Rep52 cassettes attached laterally, and an expression cassette for the human PGK promoter-driven blast fungicide resistance gene (BSD) (see [link to documentation]). Figure 5A – Figure 5P A summary of the designs for inducible promoters of the Rep gene is shown in Table 1.

[0198] Table 1

[0199]

[0200] The sequence of the PB-hiRep-1# (11,866 bp) vector is provided below:

[0201] PB-hiRep-1# (11,866 bp) (SEQ ID NO: 14)

[0202]

[0203] The sequence of the PB-hiRep-2# vector is provided below:

[0204] PB-hiRep-2# (11,861 bp) (SEQ ID NO: 15)

[0205]

[0206] The sequence of the PB-hiRep-3# vector is provided below:

[0207] PB-hiRep-3# (11,580 bp) (SEQ ID NO: 16)

[0208]

[0209] The sequence of the PB-hiRep-4# vector is provided below:

[0210] PB-hiRep-4# (11,702 bp) (SEQ ID NO: 17)

[0211]

[0212] The sequence of the PB-hiRep-5# vector is provided below:

[0213] PB-hiRep-5# (11,865 bp) (SEQ ID NO: 18)

[0214]

[0215] The sequence of the PB-hiRep-6# vector is provided below:

[0216] PB-hiRep-6# (11,860 bp) (SEQ ID NO: 19)

[0217]

[0218] The sequence of the PB-hiRep-7# vector is provided below:

[0219] PB-hiRep-7# (11,579 bp) (SEQ ID NO: 20)

[0220]

[0221] The sequence of the PB-hiRep-8# vector is provided below:

[0222] PB-hiRep-8# (11,701 bp) (SEQ ID NO: 21)

[0223]

[0224] The sequence of the PB-hiRep-9# vector is provided below:

[0225] PB-hiRep-9# (11, 861 bp) (SEQ ID NO: 22)

[0226]

[0227] The sequence of the PB-hiRep-10# vector is provided below:

[0228] PB-hiRep-10# (11,580 bp) (SEQ ID NO: 23)

[0229]

[0230] The sequence of the PB-hiRep-11# vector is provided below:

[0231] PB-hiRep-11# (11,702 bp) (SEQ ID NO: 24)

[0232]

[0233] The sequence of the PB-hiRep-12# vector is provided below:

[0234] PB-hiRep-12# (11,589 bp) (SEQ ID NO: 25)

[0235]

[0236] The sequence of the PB-hiRep-13# vector is provided below:

[0237] PB-hiRep-13# (11, 854 bp) (SEQ ID NO: 26)

[0238]

[0239] The sequence of the PB-hiRep-14# vector is provided below:

[0240] PB-hiRep-14# (11,573 bp) (SEQ ID NO: 27)

[0241]

[0242] The sequence of the PB-hiRep-15# vector is provided below:

[0243] PB-hiRep-15# (11,695 bp) (SEQ ID NO: 28)

[0244]

[0245] The sequence of the PB-hiRep-16# vector is provided below:

[0246] PB-hiRep-16# (11,421 bp) (SEQ ID NO: 29)

[0247]

[0248] Example 4: Design and validation of inducible Cap gene and target gene

[0249] Two strategies were implemented for inducible Cap gene expression. In the first strategy, the Cap gene was split into two boxes, one for VP1 expression and the other for VP2 and VP3. To reduce DNA sequence homology between VP1 and VP2 / 3 genes to improve vector stability, the DNA sequence of the VP1 coding region was optimized for human cell codon usage without altering the protein sequence. To maintain the protein-coding sequence of membrane-associated accessory proteins (MAAP), the first 439 bp of the VP1 coding region (containing the MAAP gene) was excluded from codon optimization. The DNA sequence identity between the optimized VP1 and the original VP2 / 3 genes was reduced to 80.7%.

[0250] Transgenic expression cassettes laterally attached to AAV ITRs, such as the GFP gene driven by the CMV promoter, are also included in the vector as representatives of the target gene (GOI) inserted into the vector. Additionally, a third antibiotic resistance gene against hygromycin driven by the human PGK promoter is included. All gene cassettes are subcloned into the piggyBac transposon vector and laterally attached to a core insulator.

[0251] In the PB-iCapsplit-ITRGFP-hygro-1 vector, the previously described inducible CMV promoter drives VP1 gene expression; this promoter does not contain a Kozak sequence preceding the ATG start codon of VP1. The same promoter is also used for VP2 and VP3 gene expression, which are expressed via the natural leader sequence (ggcgctaag) preceding the original ACG start codon (see [link to original text]). Figure 6A In the PB-iCapsplit-ITRGFP-hygro-2 vector, the VP1 cassette is modified with the natural VP2 leader sequence followed by an ACG start codon, and there are no other changes compared to the PB-iCapsplit-ITRGFP-hygro-1 vector (see [link to PB-iCapsplit-ITRGFP-hygro-2 vector]). Figure 6B In the PB-iCapsplit-ITRGFP-hygro-3 vector, the VP1 cassette promoter was converted to an inducible UbC promoter, and a Kozak sequence was added before the ATG start codon of the VP1 gene. No other changes were made compared to the PB-iCapsplit-ITRGFP-hygro-1 vector (see [link to PB-iCapsplit-ITRGFP-hygro-3 vector]). Figure 6CTo enhance VP1 protein expression, an 814 bp UbC intron, identified as an enhancer, was inserted before the inducible UbC promoter in the PB-iCapsplit-ITRGFP-hygro-3 vector (Bianchi et al., “A potent enhancer element in the 5′-UTR intron is crucial fortranscriptional regulation of the human ubiquitin C gene,” Gene 448(1):88-101(2009), resulting in the PB-iCapsplit-ITRGFP-hygro-4 vector (see [link to PB-iCapsplit-ITRGFP-hygro-3 vector]). Figure 6D ).

[0252] In the second strategy, the VP1, VP2, and VP3 genes retain their original architecture within a single native DNA sequence. A strongly inducible CMV promoter is applied to drive Cap gene expression. To enhance VP protein expression, the original suboptimal splicing donor site of the VP gene is optimized to a conserved donor sequence (caggtaAGT from 2309 bp to 2317 bp) without affecting the VP protein ratio (Farris and Pintel, “Improved splicing of Adeno-associated viral (AAV)capsid protein-supplying pre-mRNAs leads to increased recombinant AAV vector production,” Hum Gene Ther. 19(12): 1421–1427 (2008). The pPBBG-iCap8cd-ITRGFP-hygro vector is shown in... Figure 6E middle.

[0253] The sequence of the PB-iCap8split-ITRGFP-hygro-1 vector is provided below:

[0254] PB-iCap8split-ITRGFP-hygro-1 (17,121 bp) (SEQ ID NO: 30)

[0255]

[0256] The sequence of the PB-iCap8split-ITRGFP-hygro-2 vector is provided below:

[0257] PB-iCap8split-ITRGFP-hygro-2 (17,119 bp) (SEQ ID NO: 31)

[0258]

[0259] The sequence of the PB-iCap8split-ITRGFP-hygro-3 vector is provided below:

[0260] PB-iCap8split-ITRGFP-hygro-3 (16,835 bp) (SEQ ID NO: 32)

[0261]

[0262] The sequence of the PB-iCap8split-ITRGFP-hygro-4 vector is provided below:

[0263] PB-iCap8split-ITRGFP-hygro-4 (17,649 bp) (SEQ ID NO: 33)

[0264]

[0265] The sequence of the pPBBG-iCap8cd-ITRGFP-hygro vector is provided below:

[0266] pPBBG-iCap8cd-ITRGFP-hygro (14,262 bp) (SEQ ID NO: 34)

[0267]

[0268] Example 5: Performance of AAV Stable Production Cell Lines

[0269] To generate stable AAV-producing cell lines, one transposon from each of the iHelper, iRep, and iCap-GOI groups was used. These three transposon plasmids were co-transfected with mRNA encoding the superpiggyBac transposase (SPB-100), integrating into the genome of the stable HEK293-TetR-KRAB C20 cell line via a "cut-and-paste" mechanism. After transfection, three antibiotics, including puromycin, blastomycin, and hygromycin, were added to the cell culture medium to select cells with transposon integration. After stable cell pool generation and selection, a high-yielding cell pool was selected to monitor the long-term stability of AAV production. The suspension pool was left untreated or induced with doxycycline for three months from passage 5 to passage 25. Figure 7 As shown, the productivity of the polyclonal pool of AAV production cell lines remained stable for 25 generations under Dox induction. AAV production without Dox induction showed extremely low AAV background / leakage levels.

[0270] To evaluate the performance of stable AAV production cell lines, multiple single-cell clones were isolated and amplified for testing via single-cell printing. First, viral protein expression was assessed by Western blot analysis. The top three clones (clones A, B, and C) were used for AAV production either uninduced or induced with doxycycline. Whole-cell lysates were harvested three days after induction and prepared for Western blot analysis. Whole-cell lysates generated from triple plasmid transfection were used as positive controls. Results showed that in these cloned AAV production cell lines, the expression of helper genes, Rep genes, and Cap genes was robustly induced at levels similar to or higher than the control samples, while uninduced cells showed undetectable levels of leaky protein expression (see [link to study]). Figure 8 ).

[0271] AAV production rates of the top three clones expressing green fluorescent protein (GFP) (the target gene) were examined in a 3-liter single-use bioreactor. AAV genomic titers, determined by ddPCR titer assays, showed that AAV production was strongly induced in all clones at harvest on day 5, ranging from 4.3 × 10⁻⁶. 10 vg / mL up to 2.8×10 11 vg / mL, which is comparable to or higher than the AAV productivity using conventional triple transfection methods (see vg / mL). Figure 9 ).

[0272] The productivity of GFP-expressing clones A, B, and C was further compared with that of three representative clones (clones 1, 2, and 3) under uninduced or doxycycline-induced conditions. Clones 1, 2, and 3 contain the nucleic acid sequences disclosed in U.S. Patent No. 11,739,347 B2, which were designed using the E2A and E4 genes controlled by the inducible CMV promoter, the VA RNA controlled by the H1 promoter, and the Rep and Cap genes controlled by the native promoter. Following Dox induction, the AAV productivity of clones A, B, and C was significantly higher than that of clones 1, 2, and 3 (see [link to Dox induction]). Figure 10 ).

[0273] The functionality of AAV produced by the bioreactor was tested using a cell-based infectivity and potency assay. Purified AAV was serially diluted and transduced into HT1080 cells in 96-well plates. The percentage of GFP-positive cells in the transduced AAV was quantified by flow cytometry and used to calculate transduction units (TUs). Normalized viral genome results per TU indicated that AAV produced by the AAV-producing cell line was functional in infecting cells and expressing target genes such as GFP (see [link to relevant documentation]). Figure 11 ).

[0274] It will be apparent to those skilled in the art that other suitable modifications and adjustments can be made to the methods and applications described herein without departing from the scope of any of the implementation schemes.

[0275] It should be understood that while certain embodiments have been described and illustrated herein, the claims are not limited to the specific form or arrangement of the portions described and shown. Illustrative embodiments have been disclosed in this specification, and although specific terminology has been used, it is for general and descriptive purposes only and not for limiting purposes. In light of the above teachings, modifications and variations to the embodiments are possible. Therefore, it should be understood that the embodiments may be practiced in ways different from those specifically described.

[0276] All references cited in this article, including patents, patent applications, papers, textbooks, and other references cited in this article, if not already included, are hereby incorporated in their entirety by way of citation.

Claims

1. An isolated nucleic acid molecule operatively comprising: a. The first inducible promoter and two tetracycline operon sequences (TetO2 sequence); b. The E2A gene controlled by the first inducible promoter; c. Virus-associated (VA) noncoding RNA controlled by the second inducible promoter; d. The third inducible promoter and the TetO2 sequence; e. The E4 gene controlled by the third inducible promoter; and f. Antibiotic resistance genes.

2. The isolated nucleic acid molecule of claim 1, wherein the first inducible promoter is natural for the E2A gene.

3. The nucleic acid molecule of claim 1, wherein the second inducible promoter is a natural promoter of the VA noncoding RNA.

4. The nucleic acid molecule of claim 1, wherein the second inducible promoter is the H1 promoter.

5. The isolated nucleic acid molecule of claim 1, wherein the third inducible promoter is natural for the E4 gene.

6. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the E4 gene and the antibiotic resistance gene.

7. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeat (ITR) sequences at both the 5' and 3' ends, and wherein the nucleic acid sequence comprises a core insulator sequence inserted downstream of the 3' end ITR and upstream of the 5' end ITR.

8. An isolated nucleic acid molecule operatively comprising: a. First inducible promoter and TetO2 sequence; b. The E2A gene controlled by the first inducible promoter; c. Virus-associated (VA) noncoding RNA controlled by the second inducible promoter; d. The third inducible promoter and the TetO2 sequence; e. The E4 gene controlled by the third inducible promoter; f. An expression cassette encoding a protein ortholog and peptide tag of the VA RNA, controlled by a repressed promoter comprising a human cytomegalovirus (CMV) promoter and a TetO2 sequence; g. Termination sequence; and h. Antibiotic resistance gene.

9. The isolated nucleic acid molecule of claim 8, wherein the first inducible promoter is natural for the E2A gene.

10. The nucleic acid molecule of claim 8, wherein the second inducible promoter is a natural promoter of the VA noncoding RNA.

11. The nucleic acid molecule of claim 8, wherein the second inducible promoter is the H1 promoter.

12. The isolated nucleic acid molecule of claim 8, wherein the third inducible promoter is natural for the E4 gene.

13. The isolated nucleic acid molecule of claim 8, wherein the protein orthologs are influenza nonstructural protein 1 (NS1), Ebola virus protein 35 (VP35), orthoreovirus σ3 protein (σ3), group C rotavirus nonstructural protein 3 (NSP3), vaccinia virus E3L protein (E3L), herpes simplex virus type 1 US11 protein (US11), Epstein-Barr virus SM protein (SM), baculovirus PK2 protein (PK2), hepatitis C virus nonstructural protein 5A protein (NS5A), human herpesvirus-8 protein vIRF-2 (vIRF-2), human immunodeficiency virus protein Tat, vaccinia virus K3L protein (K3L), herpes simplex virus protein γ134.5 / ICP34.5, and human papillomavirus-18 E6 protein (E6).

14. The nucleic acid molecule of claim 8, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the E4 gene and the expression cassette.

15. The nucleic acid molecule of claim 8, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeats (ITRs) at both the 5' and 3' ends, and wherein the nucleic acid sequence comprises a core insulator sequence inserted downstream of the 3' end ITR and upstream of the 5' end ITR.

16. An isolated nucleic acid molecule operatively comprising a plasmid encoding the following elements: a. First inducible promoter and TetO2 sequence; b. The Rep78 gene contains a silenced p19 promoter located in the coding region of the Rep78 gene, wherein the silenced p19 promoter contains mutations at the SP1, TATA-1 and / or TATA-2 sites; c. The second inducible promoter and the TetO2 sequence; d. The Rep52 gene controlled by the second inducible promoter; e. Termination sequence; and f. Antibiotic resistance genes.

17. The isolated nucleic acid molecule of claim 16, wherein the first inducible promoter is a human cytomegalovirus (CMV) immediate early enhancer promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

18. The isolated nucleic acid molecule of claim 16, wherein the second inducible promoter is a human CMV immediate early enhancer-promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

19. The nucleic acid molecule of claim 16, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the termination sequence and the antibiotic resistance gene.

20. The nucleic acid molecule of claim 16, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeats (ITRs) at both the 5' and 3' ends, and wherein the nucleic acid sequence comprises a core insulator sequence inserted downstream of the 3' end ITR and upstream of the 5' end ITR.

21. An isolated nucleic acid molecule, operably comprising: a. First inducible promoter and TetO2 sequence; b. VP1 gene; c. The second inducible promoter and the TetO2 sequence; d. VP2 and VP3 genes; and e. Antibiotic resistance genes.

22. The isolated nucleic acid molecule of claim 21, wherein the first inducible promoter is a human cytomegalovirus (CMV) immediate early enhancer promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

23. The isolated nucleic acid molecule of claim 21, wherein the second inducible promoter is a human CMV immediate early enhancer-promoter, a chicken β-actin promoter (CAG) fused with a CMV immediate early enhancer, a human ubiquitin C (UbC) promoter, or a Rous sarcoma virus (RSV) promoter.

24. The isolated nucleic acid molecule of claim 21, further comprising a target gene (GOI) located downstream of the VP3 gene and upstream of the antibiotic resistance gene.

25. The isolated nucleic acid molecule of claim 24, wherein the nucleic acid molecule comprises a core insulator sequence inserted between the VP3 gene and the GOI and between the GOI and the antibiotic resistance gene.

26. The nucleic acid molecule of claim 21, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the VP1 gene and the second inducible promoter and the TetO2 sequence.

27. The nucleic acid molecule of claim 21, wherein the nucleic acid sequence comprises a core insulator sequence inserted between the VP2 and VP3 genes in d) and the antibiotic resistance gene in e).

28. The nucleic acid molecule of claim 21, wherein the nucleic acid sequence is side-attached with transposon-specific inverted terminal repeats (ITRs) at both the 5' and 3' ends, and wherein the nucleic acid sequence comprises a core insulator sequence inserted downstream of the 3' end ITR and upstream of the 5' end ITR.

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