Automated production method of viral vectors

Abstract

To provide an automated production method of virus vectors utilizing engineered viral vector-producing cell lines within a fully-enclosed cell engineering system.SOLUTION: An automated production method of virus vectors comprises the steps of: introducing an engineered mammalian viral producer cell into a fully enclosed cell engineering system, where the engineering mammalian viral producer cell includes integrated into its genome, genes under control of first and second derepressible promoters, a non-coding RNA under control of a third derepressible promoter, and a repressor element of the first, second and third derepressible promoters; transducing the mammalian viral producer cell with a vector encoding a gene of interest to produce a transduced viral producer cell;treating the mammalian viral producer cell with a binding partner of the repressor element; activating the first, second and third derepressible promoters; expanding the transduced viral producer cell and producing the viral vector within the transduced viral producer cell; and isolating the viral vector.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure provides a method for automated production of viral vectors utilizing engineered viral vector-producing cell lines in a fully enclosed cell engineering system. Exemplary viral vectors that can be produced include lentiviral vectors, adeno-associated viral vectors, baculoviral vectors, and retroviral vectors. [Background technology]

[0002] Viral vectors are of great importance both as basic research tools and for use in gene therapy. For example, adeno-associated virus (AAV) is an excellent viral vector for human gene therapy due to its safety profile and long-term expression capability. Similarly, lentiviral vectors are one of the most commonly used delivery methods in the field of gene and cell therapy. However, traditional means of producing viral vectors are mostly expensive, time-consuming, and cumbersome. Furthermore, methods that rely on crosslinking platforms (such as AAV) or multiple rounds of transient transfection (such as lentivirus) may result in vector yields that are too low or require too large amounts of plasmid DNA to accommodate most therapeutic applications. In addition, large batch processes may not be required or desirable for small-scale viral vector production.

[0003] The advantages of automating the production of viral vectors include labor time savings associated with the use of automation, as well as improved product consistency, reduced room classification, reduced cleanroom footprint, reduced complex training, and improved scale and logistics tracking. Additionally, the software can be used to streamline the documentation process by providing a history of all processing equipment, reagents, operator identification, in-process sensor data, etc., using automatically generated electronic batch records.

[0004] An automated, self-contained system for optimal production of viral vectors in engineered mammalian cells would revolutionize the field of gene therapy. There is an urgent need for technologies that allow controlled viral production for large- or small-scale mass production, providing reproducible and consistent results while limiting contamination and reducing costs.

[0005] [Summary of the Invention] In some embodiments, provided herein is a method for automated production of adeno-associated virus (AAV) viral vectors, the method comprising introducing an artificial mammalian AAV viral producer cell into a fully enclosed cell engineering system, the artificial mammalian AAV viral producer cell containing, integrated into its genome, adenovirus helper genes including E2A and E4Orf6 genes under control of a first derepressed promoter, AAV genes including Rep and Cap genes under control of a second derepressed promoter, virus-associated non-coding RNA under control of a third derepressed promoter, and a first the first, second, and third derepression promoters comprising repression elements; transducing a mammalian AAV viral producer cell with the vector encoding a gene of interest to produce a transduced viral producer cell; treating the mammalian AAV viral producer cell with a binding partner of the repression elements; activating the first, second, and third derepression promoters; growing the transduced viral producer cell and producing an AAV viral vector in the transduced viral producer cell; and isolating the viral vector, wherein the steps are performed in a closed, automated process.

[0006] Also provided herein is a method for automated production of adeno-associated virus (AAV) viral vectors, comprising: transducing a mammalian cell stably expressing one or more nucleic acids encoding TetR and / or TetR-KRAB with a first nucleic acid encoding adenoviral helper genes including E2A gene, E4Orf gene, and virus-associated non-coding RNA under control of a first derepressed promoter, a second nucleic acid encoding AAV genes including Rep and Cap genes under control of a second derepressed promoter, and optionally a third nucleic acid encoding a gene of interest under control of a third derepressed promoter; treating the mammalian cell with a binding partner of TetR; activating the first, second, and third derepressed promoters; growing the transduced cells and producing the AAV viral vector in the transduced cells; and isolating the viral vector, wherein the steps are performed in a closed, automated process.

[0007] In a further embodiment, there is provided herein a method for automated production of lentiviral vectors, the method comprising introducing an artificial mammalian lentiviral vector producing cell into a fully enclosed cell engineering system, the artificial mammalian lentiviral vector producing cell containing integrated into its genome a regulator of lentiviral virion protein expression (REV) gene under control of a first promoter, a lentiviral envelope gene under control of a second promoter, and a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under control of a third promoter. the nucleic acid sequence comprising a gene of interest, the nucleic acid sequence being flanked at both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination; transducing a mammalian lentiviral vector producing cell with the vector encoding the gene of interest to produce a transduced virus producing cell; activating the first, second, and third promoters; growing the transduced virus producing cell and producing the lentiviral vector in the transduced virus producing cell; and isolating the viral vector, wherein (a)-(e) are performed in a closed, automated process.

[0008] In a further embodiment, a method for automated production of lentiviral vectors is provided, comprising: introducing mammalian cells into a fully enclosed cell engineering system; transducing the mammalian cells with a first nucleic acid encoding a regulator of lentiviral virion protein expression (REV) gene under the control of a first promoter and an envelope glycoprotein gene under the control of a second promoter, a second nucleic acid encoding a gene of interest under the control of a third promoter, and a third nucleic acid encoding both a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene under the control of a fourth promoter; growing the transduced cells and producing lentiviral vectors in the transduced cells; and isolating the viral vectors, wherein the steps are performed in a closed automated process. [Brief description of the drawings]

[0009] [Figure 1] 1 shows a closed automated system useful for producing viral vectors as described in embodiments herein. [Diagram 2] 1 illustrates a lab space including an exemplary closed automated system useful for the production of viral vectors, as described in embodiments herein. [Diagram 3] 1 shows a flow chart of a viral vector production process that can be carried out in a closed automated system, as described in embodiments herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] When used in conjunction with the term "comprising" in the claims and / or this specification, the use of the words "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0011] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the method / device being employed to determine the value. Typically, the term is meant to include a variation of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0012] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or."

[0013] As used in this specification and claim(s), the words "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, system, host cell, expression vector, and / or composition of the invention. Additionally, the compositions, systems, cells, and / or nucleic acids of the invention can be used to achieve any of the methods described herein.

[0014] In embodiments, provided herein are methods for the automated production of viral vectors. The automated methods described herein are preferably performed in a fully enclosed cell engineering system.

[0015] As described herein, a "fully closed cell engineering system" refers to a closed system preferably including multiple chambers, and each of the steps of the various methods described herein is performed in the same or different ones of the multiple chambers of the cell engineering system. Preferably, each of the various cells, vectors, and cell culture media is contained in a different one of the multiple chambers prior to initiating the method. The cell engineering system preferably includes one or more chambers maintained at a temperature for growing cells (e.g., about 37°C), and at least one of the multiple chambers is maintained at a refrigerated temperature (e.g., about 4-8°C). "Fully closed" preferably refers to multiple chambers interconnected, including via various tubing or other fluid connection paths and connections to maintain the cleanliness and suitable sterility of the fully closed system.

[0016] As described herein, in embodiments, the methods provided utilize the COCOON platform (Octane Biotech, Kingston, Ontario), which integrates multiple unit operations in a single turnkey platform. To provide efficient and effective automated translation, the methods described utilize the concept of application-specific / sponsor-specific disposable cassettes that combine multiple unit operations, all focused on the core requirements of viral vector production. An exemplary fully enclosed cell engineering system is described in U.S. Patent Application Publication No. 2019 / 0169572, the disclosure of which is incorporated herein by reference in its entirety. An exemplary fully enclosed cell engineering system useful in the methods described herein is shown in FIG. 1. FIG. 2 illustrates a lab space including an exemplary closed automation system useful for the production of viral vectors in a high throughput arrangement, as described in embodiments herein. In embodiments, each of the closed automation systems is capable of producing a separate and unique viral vector.

[0017] In embodiments, the methods described herein are performed in a cassette of a fully enclosed cell engineering system, which may include a low temperature chamber for storage of cell culture medium, a high temperature chamber for carrying out processes involved in the production of viral vectors, the high temperature chamber being separated from the low temperature chamber by a thermal barrier and including a cell culture chamber, and one or more fluid pathways connected to the cell culture chamber, the fluid pathways providing recirculation to the cell culture chamber, removal of waste products, and homogenous gas exchange and distribution of nutrients without disturbing the cells in the cell culture chamber.

[0018] FIG. 3 shows a flow diagram of a viral vector production process that may be performed in a closed automated system, as described in embodiments herein, illustrating the various steps that may be performed within the cartridge of the closed automated system.

[0019] In an embodiment, the automated production method preferably involves introducing artificial virus-producing cells into a fully enclosed cell engineering system.

[0020] As referred to herein, an "artificial virus-producing cell" utilized in the present methods is a cell that preferably contains one or more nucleic acid molecules encoding helper genes or expression systems that enable the production of a viral vector.

[0021] As referred to in this specification, the word "introducing" may mean adding artificial virus-producing cells to one of a plurality of chambers, or may refer to the presence of artificial virus-producing cells in the cassette prior to initiating the method.

[0022] In an embodiment, the method described herein is configured to perform multiple rounds of one or more of feeding, washing, and monitoring of artificial virus-producing cells. These various actions may be performed in any order, and may be performed alone or in combination with another action. In an embodiment, concentrating the cells includes centrifugation, removal of supernatant after precipitation, or filtration. Preferably, the optimization process further includes adjusting the parameters of centrifugation or filtration in a self-regulating process.

[0023] The methods described herein suitably further comprise transducing the artificial virus-producing cell with a vector encoding a gene of interest to produce a transduced virus-producing cell.

[0024] As referred to herein, "transduction" or "transducing" refers to the introduction of an exogenous nucleic acid molecule, including a vector, into a cell. A "transduced" cell contains an exogenous nucleic acid molecule within the cell, which induces a phenotypic change in the cell. The transduced nucleic acid molecule may be integrated into the genomic DNA of the host cell and / or may be maintained by the cell transiently or extrachromosomally long term. A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant," "transduced," "transfected," or "transgenic" organism. Several transduction and transfection techniques are commonly known in the art. See, e.g., Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:197 (1981). Transduction can include using transfection systems such as liposome-, lipid-, or polymer-based systems, and can also include using mechanical transfection such as a gene gun, electroporation, etc.

[0025] As used herein, a "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which a nucleic acid molecule described herein may be attached, resulting in the replication and / or expression of the attached nucleic acid molecule in a cell. "Vector" includes episomal (e.g., plasmid) and non-episomal vectors. The term "vector" includes both viral and non-viral means for introducing a nucleic acid molecule into a cell in vitro, in vivo, or ex vivo. The term vector may also include synthetic vectors. Vectors may be introduced into a desired host cell by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection. Vectors may include various regulatory elements, including promoters.

[0026] A "viral vector" produced by the methods described herein suitably refers to a product virus that can be used to introduce a nucleic acid molecule into a cell in vitro, in vivo, or ex vivo for therapeutic or industrial purposes. The viral vectors produced by the various methods described herein can be harvested or isolated and stored until the final desired application.

[0027] "Gene" refers to an assembly of nucleotides that encodes a polypeptide and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can function as a regulatory sequence before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. In some embodiments, the gene is integrated with multiple copies. In some embodiments, the gene is integrated with a predetermined copy number.

[0028] As referred to herein, the term "gene of interest" or "GOI" is used to describe a heterologous gene. As referred to herein, the term "heterologous gene" or "HG" in relation to a nucleic acid sequence, such as a coding sequence or a control sequence, refers to a nucleic acid sequence, such as a gene, that is not normally combined together and / or is not normally associated with a particular cell. In some embodiments, a heterologous gene is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with different codons than the natural gene). Allelic variation or naturally occurring mutation events do not give rise to heterologous DNA as used herein.

[0029] Preferably, the gene of interest is a gene of therapeutic interest. As used herein, "gene of therapeutic interest" refers to any functionally related nucleotide sequence. Thus, the gene of therapeutic interest of the present disclosure may include any desired gene that encodes a protein that is defective or missing from the genome of a target cell, or encodes a non-native protein that has a desired biological or therapeutic effect (e.g., antiviral function), or a sequence that may correspond to a molecule with antisense or ribozyme function. Representative (non-limiting) examples of suitable genes of therapeutic interest include those used to treat inflammatory, autoimmune, chronic and infectious diseases, including diseases such as AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, various anemias, thalassemia, and various blood diseases including hemophilia, genetic defects such as cystic fibrosis, Gaucher's disease, adenosine deaminase (ADA) deficiency, emphysema, etc. A number of antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of an mRNA) useful for antisense therapy of cancer and viral diseases have been described in the art and are examples of suitable therapeutic target genes.

[0030] In an exemplary embodiment, the methods described herein, and thus the therapeutic gene, are useful for producing viral vectors for ultra-rare disease applications. Although such diseases may not require large amounts of viral vectors (as in the case of one or a few patients, or tens of patients, or hundreds of patients needing treatment), sterility, reproducibility, and process control are crucial in such applications. The methods described herein utilize closed automated systems, allowing for desired levels of production control.

[0031] In embodiments, the method further comprises growing the transduced virus-producing cells and producing the viral vectors in the transduced virus-producing cells. As described herein, the method of growing the transduced virus-producing cells preferably comprises at least one or more of feeding, washing, and monitoring. "Growing" the transduced virus-producing cells refers to various methods that allow the cells to grow until they reach a predetermined desired culture size. The predetermined culture size may include a sufficient number of cells to allow the production of a suitable or desired number of viral vectors. In some embodiments, the number of viral-producing cells is about 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, approximately 10 8 cells, approximately 10 9 cells, or approximately 10 10 Each cell is a single cell.

[0032] The production methods described herein further suitably include isolating the viral vector produced in the transduced viral-producing cells. Methods of isolating or otherwise harvesting the viral vector include harvesting the transduced and grown viral-producing cells, and may include lysing or otherwise disrupting the cell membrane and harvesting the viral vector.

[0033] Also, the isolation of viral vectors may include additional steps such as centrifuging or filtering virus-producing cells, washing cells, or filtering isolated viral vectors. Isolation preferably includes various separation techniques that may rely on filtration, centrifugation, gel purification techniques, gradient separation techniques, etc. The viral vectors can be removed by washing, or by binding the vectors to a surface (e.g., a surface coated with retronectin or fibronectin) and then transferring the cells to a different chamber.

[0034] As described herein, the elements of the automated production method are preferably performed in a closed automated process. The term "closed" process preferably refers to the use of a cartridge or other containment system that does not allow interaction with the outside environment (unless desired), and is preferably a sterile process. An "automated" process, or "automation" of a process, refers to the control of one or more processes of a cell engineering system by external control, including a microprocessor for monitoring and altering parameters based on defined or pre-set conditions or desired characteristics.

[0035] In embodiments, the closed automated process is a self-regulating process. That is, a closed automated process is a process that does not require input from an external (human) user and may determine, through various computer programs and conditions, necessary modifications to the cell culture or other characteristics to optimize the automated process. In embodiments, the closed automated process includes monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor. As described herein, the use of these various sensors in a fully closed cell engineering system occurs at various times and places within the system and works together in a coordinated manner to achieve optimization. For example, the closed automated process may adjust (e.g., increase or decrease) one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density of the virus-producing cell culture based on this monitoring.

[0036] The automated process can also be based on the intrinsic characteristics of the starting cell population, including, for example, total cell number, source of cells, density of cells, age of cells, etc. These starting cell population characteristics can be input into a computer controlled system prior to initiating the automated method, and the system then makes various initial modifications, for example, lactose, oxygen and carbon dioxide concentrations, flow rates, incubation times, pH, etc., to optimize the method. Alternatively, monitoring of cell processes allows for automated characterization of the progression of cell culture sequences from the starting population, allowing conditions for optimized final cell culture characteristics to be adjusted on a case-by-case basis.

[0037] In further embodiments, the cell engineering system recycles nutrients, waste products, released cytokines, and / or dissolved gases during various method processes. This recycle helps to support the production of the desired viral vector. Other mechanisms for optimizing the production of viral vectors include modifying and controlling the flow rate of the medium provided to the cells. As the cells begin to grow, the circulation rate of the medium provided increases, improving gas exchange and allowing oxygen and carbon dioxide to enter and leave the cell culture depending on the state and requirements of the cells at that time.

[0038] In embodiments, the artificial virus-producing cells utilized in the automated method are mammalian cells. As used herein, the term "mammalian cells" includes cells derived from any member of the mammalian species, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, and the like. 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 mutants (e.g., POTELLIGENT®, Lonza, Slough, UK), CHOK1SV GS-KO (glutamine synthetase knockout) cells including all mutants (e.g., XCEED™, Lonza, Slough, UK). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK293, HEK293T, HeLa cells, or HT1080 cells.

[0039] Mammalian cells include mammalian cell cultures, which can be either adherent or suspension cultures. Adherent cultures refer to cells that grow on a substrate surface, such as a plastic surface, plate, dish, or other suitable cell culture growth platform, and can be adhesion-dependent. Suspension cultures refer to cells that can be maintained, for example, in culture flasks or large suspension tanks that allow for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize stirring or agitation mechanisms to provide adequate mixing. Media and conditions for maintaining cells in suspension are generally known in the art. Exemplary suspension cell cultures include human HEK293 clonal cells.

[0040] In embodiments, the methods for producing viral vectors provided herein produce adeno-associated viral (AAV) vectors.

[0041] As used herein, the term "adeno-associated virus (AAV) vector" refers to a small-sized, replication-deficient, non-enveloped virus that contains single-stranded DNA of the Parvoviridae and Dependoparvovirus genus. More than 10 adeno-associated virus serotypes have been identified to date, with serotype AAV2 being the best characterized. Other non-limiting examples of AAV serotypes are ANC80, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In addition to these serotypes, AAV pseudotypes have been developed. AAV pseudotypes contain the capsid of a first serotype and the genome of a second serotype (e.g., pseudotype AAV2 / 5 corresponds to AAV with the genome of serotype AAV2 and the capsid of AAV5).

[0042] As referred to herein, the term "adenovirus" refers to a non-enveloped virus with an icosahedral nucleocapsid containing double-stranded DNA of the Adenoviridae family. More than 50 adenovirus subtypes have been isolated from humans, and many additional subtypes have been isolated from other mammals and birds. See, for example, Ishibashi et al., "Adenoviruses of animals" In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, NY, pp. 497-562 (1984); Strauss, "Adenovirus infections in humans" In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, NY, pp. 451-596 (1984). These subtypes belong to the Adenoviridae family, which is currently divided into two genera: Mastadenovars and Aviadenovars. All adenoviruses are morphologically and structurally similar. However, in humans, adenoviruses exhibit different immunological properties, and are therefore classified into serotypes. Two human serotypes of adenovirus, namely AV2 and AV5, have been intensively studied and provide most of the general information about adenoviruses.

[0043] In embodiments, the methods of producing viral vectors provided herein produce lentiviral vectors.

[0044] As referred to herein, the term "lentiviral vector" refers to a small spherical enveloped virus that contains two single-stranded RNA molecules and belongs to the retroviridae family.Lentivirus contains gag, pol and env genes, and further distinguishes itself from other members of the retroviral family by having two regulatory genes, namely tat and rev.Lentiviral vector is widely known in the art as a useful tool in molecular biology for inducing the expression of target genes in cultured cells and animal tissues.

[0045] In embodiments, the methods for producing viral vectors provided herein produce retroviral vectors.

[0046] As referred to herein, the term "retrovirus" refers to one or more members of the Retroviridae family, which are small, spherical enveloped viruses that contain two single-stranded RNA molecules.Retroviruses convert their RNA molecules into DNA, which is then integrated into the host genome of infected cells.Retrovirus-based vectors are well known in the field of gene therapy for cancer treatment, and immune cells are reprogrammed to target and destroy cancer cells.

[0047] In embodiments, the methods for producing viral vectors provided herein produce baculovirus vectors.

[0048] As referred to herein, the term "baculovirus" refers to a rod-shaped virus containing circular dsDNA and one or more members of the family Baculoviridae. It is known to infect and replicate primarily in insect larvae. The baculovirus expression vector system is well established and highly useful for the production of proteins in eukaryotic cells (Summers et al., 2006).

[0049] In additional embodiments, the method preferably utilizes insect cells as virus production cells. As referred to herein, "insect cells" preferably refers to, but are not limited to, cells derived from insects such as members of the order Lepidoptera that are used for the expression and manufacture of proteins and / or baculovirus vector production.

[0050] In embodiments, the method preferably utilizes Sf9 cells. As referred to herein, "Sf9 cells" are an insect cell line derived from the pupal ovary tissue of the worm Spodoptera frugiperda, commonly used for the expression and manufacture of proteins and / or baculoviral vector production.

[0051] In embodiments, the amount of viral vector produced by the methods described herein is at least about 10 10 For example, the amount of viral vector produced by the methods described herein is at least about 10 10 viral vectors, or at least about 10 11 viral vectors, or at least about 10 12 viral vectors, or at least about 10 13 viral vectors, or at least about 10 14 viral vectors, or approximately 10 10 ~10 14 viral vectors, or approximately 10 10 ~10 13 viral vectors, or approximately 10 10 ~10 12 viral vectors, or approximately 10 10 pieces, about 10 11 pieces, about 10 12 Pieces, or about 10 13 It is a viral vector.

[0052] In an embodiment, the method described herein is for the production of adeno-associated virus (AAV) virus vectors. Such a process preferably comprises introducing an artificial mammalian AAV virus production cell into a fully enclosed cell engineering system. The AAV virus production cell preferably comprises adenovirus helper genes, including E2A and E4Orf6 genes, integrated into its genome under the control of a first derepressed promoter.

[0053] Exemplary artificial mammalian AAV virus-producing cells suitable for use in the methods of producing AAV viral vectors are described in detail in U.S. Provisional Patent Application No. 62 / 783,589, filed December 21, 2018, U.S. Provisional Patent Application No. 62 / 866,092, filed June 25, 2019, and U.S. Nonprovisional Patent Application No. 16 / 719,251, filed December 18, 2019, the disclosures of each of which are incorporated by reference in their entireties herein.

[0054] As described herein, the mammalian AAV virus-producing cells utilized in the present method preferably contain nucleic acid molecules encoding viral helper genes. The viral helper genes include various adenovirus genes, herpesvirus genes, and bocavirus genes (see, e.g., Guido et al., "Human bocavirus: Current knowledge and future challenges", World J. Gastroenterol. 22:8684-8697, the disclosure of which is incorporated herein by reference in its entirety). In an exemplary embodiment, the viral helper genes are adenovirus helper genes. As referred to herein, the term "adenovirus helper genes" or "AV helper genes" refers to genes consisting of one or more nucleic acid sequences derived from one or more adenovirus subtypes or serotypes that contribute to adeno-associated virus replication and packaging. In some embodiments, the adenovirus helper genes are E1A, E1B, E2A, E4 (including E4Orf6), VA, or combinations thereof, or other adenovirus helper genes. In an exemplary embodiment, the adenovirus helper gene comprises both E2A and E4Orf6 genes.Preferably, an internal ribosome entry site (IRES) element is comprised between E2A and E4Orf6 genes.The IRES element initiates the translation of E4Orf6 gene after E2A gene in a single expression cassette and provides stability to the construct.

[0055] Preferably, the promoter used to control the transcription of various genes for producing AAV viral vector is a derepression promoter.As used herein, "derepression promoter" refers to a structure that includes a functional promoter and additional elements or sequences that can be combined with the repression element to cause the repression of the functional promoter."Repression" refers to the reduction or inhibition of the initiation of transcription of downstream coding or non-coding gene sequence by the promoter.

[0056] In a further embodiment, the method for automated production of AAV viral vectors includes an artificial mammalian virus producer cell containing AAV genes including the Rep and Cap genes under the control of a second derepressed promoter.

[0057] As referred to herein, the term "Rep" gene refers to the art-recognized region of the AAV genome that encodes viral replication proteins collectively required to replicate the viral genome, or functional homologues thereof, such as the human herpesvirus 6 (HHV-6) rep gene, which is also known to mediate AAV-2 DNA replication. Thus, the rep coding region can include genes encoding AAV Rep78 and Rep68 ("long form Rep"), and Rep52 and Rep40 ("short form Rep"), or functional homologues thereof. The Rep coding region, as used herein, can be derived from any viral serotype, such as the AAV serotypes described herein. The region need not contain all of the wild-type genes, but can be modified (e.g., by nucleotide insertion, deletion, or substitution), so long as there is a rep gene present that provides sufficient integration function when expressed in a suitable target cell. See, e.g., Muzyczka, N., Current Topics in Microbiol. and Immunol. 158:97-129 (1992), and Kotin, RM, Human Gene Therapy 5:793-801 (1994).

[0058] As referred to herein, the term "Cap" gene refers to the art-recognized region of the AAV genome that encodes the viral capsid proteins. Illustrative (non-limiting) examples of these capsid proteins are the AAV capsid proteins VP1, VP2, and VP3. The Cap genes used in this disclosure can be from any AAV serotype or combination of AAV serotypes.

[0059] In a further embodiment, the AAV virus producing cell may contain a virus-associated non-coding RNA under the control of a third derepressed promoter.

[0060] In a further embodiment, the method utilizes an AAV virus producing cell containing repressing elements of the first, second, and third derepressed promoters.

[0061] As referred to herein, a "repressing element" refers to a protein or polypeptide that can bind to a promoter (or near a promoter) to reduce or inhibit the activity of the promoter. The repressing element may interact with a substrate or binding partner of the repressing element such that the repressing element undergoes a conformational change. This conformational change of the repressing element removes the ability of the repressing element to reduce or inhibit the promoter, resulting in "derepression" of the promoter, thereby allowing the promoter to proceed with initiation of transcription. A "functional promoter" refers to a promoter that is capable of initiating transcription in the absence of the action of the repressing element. Various functional promoters that can be used in the practice of the present invention are known in the art, for example, P CMV , P H1 , P19, P5, P40, and promoters of adenovirus helper genes (e.g., E1A, E1B, E2A, E4Orf6, and VA).

[0062] In an embodiment, the method suitably comprises transducing a mammalian AAV virus-producing cell with a vector encoding a gene of interest to produce a transduced virus-producing cell.

[0063] In embodiments, the method further comprises treating a mammalian AAV virus-producing cell with a binding partner of the inhibitory element.

[0064] Exemplary repression elements and their corresponding binding partners that can be used as derepression promoters are known in the art and include the cumate gene-switch system (CuO operator, CymR repression element and co-binding partner) (see, e.g., 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 derepression promoter system described herein), and the TetO / TetR system described herein (see, e.g., Yao et al., "Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells," Human Gene Therapy 2002, 14:131-132, 2002). 9:1939-1950 (1998), the disclosure of which is incorporated herein by reference in its entirety.

[0065] In a further embodiment, the method comprises activating the first, second, and third derepressed promoters.

[0066] In a further embodiment, the method suitably comprises growing the transduced virus-producing cells to produce the AAV viral vector, and then isolating the viral vector.

[0067] In an embodiment, the method steps are performed in a closed automated process.

[0068] In a further embodiment, provided herein is a method for automated production of adeno-associated virus (AAV) viral vectors, comprising: transducing a mammalian cell stably expressing one or more nucleic acids encoding TetR and / or TetR-KRAB with a first nucleic acid encoding adenoviral helper genes including E2A gene, E4Orf gene, and virus-associated non-coding RNA under control of a first derepressed promoter, a second nucleic acid encoding AAV genes including Rep and Cap genes under control of a second derepressed promoter, and optionally a third nucleic acid encoding a gene of interest under control of a third derepressed promoter; treating the mammalian cell with a binding partner of TetR; activating the first, second, and third derepressed promoters; growing the transduced cells and producing the AAV viral vector in the transduced cells; and isolating the viral vector, wherein the steps are performed in a closed, automated process.

[0069] In embodiments, the cells utilized in the production methods, including the artificial mammalian AAV virus producer cells, are in some embodiments mammalian cell cultures, preferably suspension cultures. Exemplary mammalian cells include human cells, including CHO cells or HEK cells.

[0070] In a further embodiment, the method for automated production of AAV viral vectors preferably utilizes a derepressed promoter that includes a functional promoter and two tetracycline operator sequences (TetO2). In such an embodiment, the repressing element is a tetracycline repressor protein.

[0071] In an exemplary embodiment, the derepressed promoter comprises a functional promoter and two tetracycline operator sequences (TetO2), as described herein. When two tetracycline repressor proteins (TetR (the repressor element of the TetO2 sequence)) are bound to the TetO2 sequence, little or no transcription occurs from these promoters. Upon binding of a binding partner of TetR (preferably doxycycline (Dox)), the TetR protein changes conformation, releases from the TetO2 sequence, and the functional promoters begin their normal transcription process as in nature. Thus, in an embodiment, the automated production method preferably further comprises treating cells with doxycycline, which activates the derepressed promoter, allowing the production of the viral vector.

[0072] In embodiments, the method for automated production of AAV viral vectors includes wherein the gene of interest is a gene of therapeutic interest.

[0073] In embodiments, the automated method for producing AAV viral vectors comprises at least about 10 10 For example, the amount of AAV viral vector produced by the methods described herein is at least about 10 10 AAV viral vectors, or at least about 10 11 AAV viral vectors, or at least about 10 12 AAV viral vectors, or at least about 10 13 AAV viral vectors, or at least about 10 14 AAV viral vectors, or approximately 10 10 ~10 14 AAV viral vectors, or approximately 10 10 ~10 13 AAV viral vectors, or approximately 10 10 ~10 12 AAV viral vectors, or approximately 10 10 pieces, about 10 11 pieces, about 10 12 Pieces, or about 10 13It is an AAV viral vector.

[0074] In an additional exemplary embodiment, a method for automated production of lentiviral vectors is disclosed, comprising introducing an artificial mammalian lentiviral vector producing cell into a fully enclosed cell engineering system. Methods for producing lentiviral vector producing cells are described in U.S. Provisional Patent Application No. 62 / 890,904, filed August 23, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0075] As used herein, a "lentiviral vector producing cell" refers to a cell that contains integrated into its genome the necessary elements to produce a lentiviral vector.

[0076] In an embodiment, the method utilizes a lentiviral vector producing cell that contains integrated into its genome a regulator of lentiviral virion protein expression (REV) gene under the control of a first promoter, a lentiviral envelope gene (envelope glycoprotein gene) under the control of a second promoter, and a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene under the control of a third promoter. In a preferred embodiment, the nucleic acid sequence is flanked at both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination.

[0077] As disclosed herein, the lentiviral regulator of virion protein expression (REV) is an RNA-binding protein that promotes late gene expression and is also important for the transport of unspliced ​​or singly spliced ​​mRNAs encoding viral structural proteins from the nucleus to the cytoplasm.

[0078] The envelope glycoprotein gene, preferably the vesicular stomatitis virus glycoprotein (VSV-G) gene, is expressed and displayed on the surface of the lentiviral vector and mediates transduction of the lentiviral vector into target cells.

[0079] GAG encodes a polyprotein translated from unspliced ​​mRNA, which is then cleaved by viral protease (PR) into matrix protein, capsid, and nucleocapsid proteins. Lentiviral polymerase (POL) is expressed as a GAG-POL polyprotein as a result of ribosomal frameshift during GAG mRNA translation, and encodes the enzyme proteins reverse transcriptase, protease, and integrase. These three proteins are associated with the viral genome within the virion. Preferably, the GAG ​​gene is the HIV GAG gene, and the POL gene is the HIV POL gene.

[0080] In a preferred embodiment, the expression cassette is flanked by transposon-specific inverted terminal repeats (ITRs) at both the 5' and 3' ends.

[0081] Exemplary promoters for use in lentiviral vector producing cells are known in the art and include derepressed promoters.Preferably, the expression cassette further encodes the repression elements of the first, second and third derepressed promoters.In an embodiment, the derepressed promoter comprises a functional promoter and a tetracycline operator sequence (TetO), and the repression element is a tetracycline repressor protein as described herein.

[0082] In a further embodiment, a method of producing a lentiviral vector comprises transducing a mammalian lentiviral vector producing cell with a vector encoding a gene of interest, in an embodiment, the gene of interest is a gene of therapeutic interest.

[0083] In a further embodiment, the method comprises activating the first, second, and third promoters in the lentiviral vector producing cells and growing the transduced virus producing cells.

[0084] In a further embodiment, the method includes suitably isolating the produced lentiviral vector. Methods for isolating the produced viral vector are described herein.

[0085] In an exemplary embodiment, the method is performed in a closed, automated process.

[0086] In a further embodiment, a method for automated production of lentiviral vectors is provided, the method comprising: introducing mammalian cells into a fully enclosed cell engineering system; transducing the mammalian cells with a first nucleic acid encoding a regulator of lentiviral virion protein expression (REV) gene under the control of a first promoter and an envelope glycoprotein gene under the control of a second promoter; a second nucleic acid encoding a gene of interest under the control of a third promoter; and a third nucleic acid encoding both a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene under the control of a fourth promoter; growing the transduced cells and producing lentiviral vectors in the transduced cells; and isolating the viral vectors, (a)-(d) being performed in a closed automated process. Methods for transient production of lentiviral vectors are described in U.S. Provisional Patent Application No. 62 / 949,848, filed December 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0087] As described herein, the automated methods preferably utilize mammalian cells, which are mammalian cell cultures, in embodiments suspension cultures. Exemplary cells include human cells, such as HEK293 or HEK293T cells.

[0088] In a further embodiment, the method utilizes a nucleic acid comprising a Krüppel-associated box (KRAB) sequence following a sequence encoding a tetracycline repressor protein.

[0089] As described herein, preferably the KRAB sequence (approximately 75 amino acids) is a transcriptional repression domain derived from human zinc finger protein 10 and increases regulation of repressor elements, preferably TetR repressor elements. The KRAB domain functions as a transcriptional repressor element when tethered to template DNA by the DNA binding domain.

[0090] In further embodiments, the methods provided herein further comprise a transposon-specific ITR that is a lepidopteran transposon (PIGGYBAC®) ITR.

[0091] Transposable elements (transposons) can move around the genome of a cell and are useful for inserting genes to produce transgenic organisms. The lepidopteran transposon PIGGYBAC® can move within the genomes of a wide variety of species and is useful as a gene transduction vector. The transposon structure contains a complex repeat organization consisting of internal repeats (IR), spacers, and terminal repeats (TR) at both ends, as well as a single open reading frame that codes for a transposase.

[0092] Exemplary lepidopteran transposon (PIGGYBAC®) ITRs that can be used in the plasmids and expression cassettes described herein include those disclosed in U.S. Pat. No. 7,105,343, the disclosure of which is incorporated herein by reference in its entirety.

[0093] In embodiments, the automated method for producing lentiviral vectors comprises at least about 10 10 For example, the amount of lentiviral vector produced by the methods described herein is at least about 10 10 lentiviral vectors, or at least about 10 11 lentiviral vectors, or at least about 10 12 lentiviral vectors, or at least about 10 13lentiviral vectors, or at least about 10 14 lentiviral vectors, or approximately 10 10 ~10 14 lentiviral vectors, or approximately 10 10 ~10 13 lentiviral vectors, or approximately 10 10 ~10 12 lentiviral vectors, or approximately 10 10 pieces, about 10 11 pieces, about 10 12 Pieces, or about 10 13 This is a lentiviral vector.

[0094] In an embodiment, the method steps are performed in a closed automated process and preferably include monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

[0095] Also provided herein is a method for treating a mammalian subject, preferably a human subject, with the AAV or lentivirus vector produced according to the various methods described herein.Preferably, the method is used to treat a human subject having a gene of interest, including a gene of interest for treatment.Administration to a human subject can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.

[0096] Additional Exemplary Embodiments Embodiment 1 is a method for automated production of adeno-associated virus (AAV) viral vectors, the method comprising introducing an artificial mammalian AAV viral producer cell into a fully enclosed cell engineering system, the artificial mammalian AAV viral producer cell containing adenovirus helper genes, including E2A and E4Orf6 genes under the control of a first derepressed promoter, AAV genes, including Rep and Cap genes under the control of a second derepressed promoter, virus-associated non-coding RNA, and a first, second, and third derepressed promoter, integrated into its genome. and a third derepression promoter, transducing a mammalian AAV viral producer cell with the vector encoding a gene of interest to produce a transduced viral producer cell, treating the mammalian AAV viral producer cell with a binding partner of the repression elements, activating the first, second, and third derepression promoters, growing the transduced viral producer cell and producing an AAV viral vector in the transduced viral producer cell, and isolating the viral vector, wherein the steps are performed in a closed, automated process.

[0097]

[0023] Embodiment 2 includes the method of embodiment 1, wherein the artificial mammalian AAV virus-producing cell is a mammalian cell culture.

[0098] Embodiment 3 includes the method of embodiment 2, wherein the mammalian cell culture is a suspension culture.

[0099]

[0023] Embodiment 4 includes the method of any one of embodiments 1 to 3, wherein the artificial mammalian AAV virus-producing cell is a Chinese Hamster Ovary (CHO) cell.

[0100] Embodiment 5 includes the method of any one of embodiments 1 to 3, wherein the artificial mammalian AAV virus-producing cell is a human cell.

[0101] Embodiment 6 includes the method of embodiment 5, wherein the human cells are human embryonic kidney (HEK) cells.

[0102] Embodiment 7 includes the method of any of embodiments 1 to 6, wherein each of the derepressed promoters comprises a functional promoter and two tetracycline operator sequences (TetO2), and the repression element is a tetracycline repressor protein.

[0103] Embodiment 8 includes the method of embodiment 7, wherein treating includes treating with doxycycline.

[0104] Embodiment 9 includes the method of any one of Embodiments 1 to 8, wherein the target gene is a gene of therapeutic interest.

[0105] Embodiment 10 is a method for producing an AAV viral vector comprising the steps of: 10 The method according to any one of embodiments 1 to 9, wherein the vector is a viral vector.

[0106] Embodiment 11 includes a method according to any of embodiments 1-10, wherein the closed automated process includes monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

[0107] Embodiment 12 includes the method of any of embodiments 1-11, wherein transducing includes viral infection, electroporation, liposomal transfection, or membrane disruption.

[0108] Embodiment 13 is a method for automated production of adeno-associated virus (AAV) viral vectors, comprising: transducing a mammalian cell stably expressing one or more nucleic acids encoding TetR and / or TetR-KRAB with a first nucleic acid encoding adenovirus helper genes including E2A gene, E4Orf gene, and virus-associated non-coding RNA under the control of a first derepressed promoter, a second nucleic acid encoding AAV genes including Rep and Cap genes under the control of a second derepressed promoter, and optionally a third nucleic acid encoding a gene of interest under the control of a third derepressed promoter; treating the mammalian cell with a binding partner of TetR; activating the first, second, and third derepressed promoters; growing the transduced cells and producing AAV viral vectors in the transduced cells; and isolating the viral vectors, wherein the steps are performed in a closed automated process.

[0109] Embodiment 14 includes the method of embodiment 13, wherein the mammalian cell is a mammalian cell culture.

[0110] Embodiment 15 includes the method of embodiment 14, wherein the mammalian cell culture is a suspension culture.

[0111] Embodiment 16 includes the method of any of embodiments 12 to 15, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0112] Embodiment 17 includes the method of any one of embodiments 12 to 15, wherein the mammalian cell is a human cell.

[0113] Embodiment 18 includes the method of embodiment 17, wherein the human cells are human embryonic kidney (HEK) cells.

[0114] Embodiment 19 includes the method of any of embodiments 12-18, wherein treating includes treating with doxycycline.

[0115] Embodiment 20 includes the method according to any one of embodiments 12 to 19, wherein the target gene is a gene of therapeutic interest.

[0116] Embodiment 21 is a method for producing an AAV viral vector comprising the steps of: 10 The method according to any one of embodiments 12 to 20, wherein the vector is a viral vector.

[0117] Embodiment 22 includes a method according to any of embodiments 12-21, wherein the closed automated process includes monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

[0118] Embodiment 23 includes the method of any of embodiments 12-22, wherein transducing includes viral infection, electroporation, liposomal transfection, or membrane disruption.

[0119] Embodiment 24 is a method for automated production of lentiviral vectors, comprising introducing an artificial mammalian lentiviral vector producing cell into a fully enclosed cell engineering system, the artificial mammalian lentiviral vector producing cell expressing a lentiviral regulator of expression of a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL), both under the control of a third promoter, integrated into its genome. ) gene, wherein the nucleic acid sequence is flanked at both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination; transducing a mammalian lentiviral vector producing cell with the vector encoding the gene of interest to produce a transduced virus producing cell; activating the first, second, and third promoters; growing the transduced virus producing cell and producing the lentiviral vector in the transduced virus producing cell; and isolating the viral vector, wherein the steps are performed in a closed, automated process.

[0120]

[0031] Embodiment 25 includes the method of embodiment 24, wherein the artificial mammalian lentiviral vector producing cell is a mammalian cell culture.

[0121] Embodiment 26 includes the method of embodiment 25, wherein the mammalian cell culture is a suspension culture.

[0122] Embodiment 27 includes the method of any one of embodiments 24 to 26, wherein the artificial mammalian lentiviral vector-producing cell is a human cell.

[0123] Embodiment 28 includes the method of embodiment 27, wherein the human cells are human embryonic kidney (HEK) cells.

[0124] Embodiment 29 includes the method of any of embodiments 24-28, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

[0125] Embodiment 30 includes the method of any one of embodiments 24 to 29, wherein the lentiviral envelope gene is a vesicular stomatitis virus glycoprotein (VSV-G) gene.

[0126] Embodiment 31 includes the method of any of embodiments 24 to 30, wherein the first promoter, the second promoter, and the third promoter are derepressed promoters.

[0127]

[0036] Embodiment 32 includes the method of embodiment 31, wherein the artificial mammalian lentiviral vector producing cell further includes integrating repression elements of the first, second, and third derepressed promoters into its genome.

[0128] Embodiment 33 includes the method of embodiment 32, wherein each of the derepressed promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repression element is a tetracycline repressor protein.

[0129] Embodiment 34 includes the method of embodiment 33, further comprising a Krüppel association box sequence following the sequence encoding the tetracycline repressor protein.

[0130] Embodiment 35 includes the method of any of embodiments 24 to 34, wherein the transposon-specific ITRs are lepidopteran transposon (PIGGYBAC®) ITRs.

[0131] Embodiment 36 includes the method of any one of Embodiments 24 to 35, wherein the target gene is a gene of therapeutic interest.

[0132] Embodiment 37 is a method for producing a lentiviral vector comprising the steps of: 10The method according to any one of embodiments 24 to 36, wherein the vector is a viral vector.

[0133] Embodiment 38 includes a method according to any of embodiments 24-37, wherein the closed automated process includes monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

[0134] Embodiment 39 includes the method of any of embodiments 24-38, wherein transducing includes viral infection, electroporation, liposomal transfection, or membrane disruption.

[0135] Embodiment 40 is a method for automated production of lentiviral vectors, comprising: introducing mammalian cells into a fully enclosed cell engineering system; transducing the mammalian cells with a first nucleic acid encoding a regulator of lentiviral virion protein expression (REV) gene under the control of a first promoter and an envelope glycoprotein gene under the control of a second promoter, a second nucleic acid encoding a gene of interest under the control of a third promoter, and a third nucleic acid encoding both a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene under the control of a fourth promoter; growing the transduced cells and producing lentiviral vectors in the transduced cells; and isolating the viral vectors, wherein the steps are performed in a closed automated process.

[0136] Embodiment 41 includes the method of embodiment 40, wherein the mammalian cell is a mammalian cell culture.

[0137] Embodiment 42 includes the method of embodiment 41, wherein the mammalian cell culture is a suspension culture.

[0138] Embodiment 43 includes the method of any one of embodiments 40 to 42, wherein the mammalian cell is a human cell.

[0139] Embodiment 44 includes the method of embodiment 43, wherein the human cells are human embryonic kidney (HEK) cells.

[0140] Embodiment 45 includes the method of any of embodiments 40-44, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

[0141] Embodiment 46 includes the method of any one of embodiments 40 to 46, wherein the envelope glycoprotein gene is a vesicular stomatitis virus glycoprotein (VSV-G) gene, and the VSV-G gene and the REV gene are codon-optimized.

[0142] Embodiment 47 includes the method of any one of embodiments 40 to 46, wherein the target gene is a gene of therapeutic interest.

[0143] Embodiment 48 is a method for producing a lentiviral vector comprising the steps of: 10 The method according to any one of embodiments 40 to 47, wherein the vector is a viral vector.

[0144] Embodiment 49 includes a method according to any of embodiments 40-48, wherein the closed automated process includes monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

[0145] Embodiment 50 includes the method of any of embodiments 40-49, wherein transducing includes viral infection, electroporation, liposomal transfection, or membrane disruption.

[0146] Embodiment 51 is an AAV viral vector produced by the method of any one of embodiments 1 to 23.

[0147] Embodiment 52 is a lentiviral vector produced by the method according to any one of embodiments 14 to 50.

[0148] Embodiment 53 is a method of treatment with an AAV vector, comprising administering the AAV vector described in embodiment 51 to a mammalian subject.

[0149] Embodiment 54 includes the method of embodiment 53, wherein administering includes inhalation, injection, or intravenous administration.

[0150] Embodiment 55 is a method of treatment with a lentiviral vector, comprising administering to a mammalian subject the lentiviral vector of claim 52.

[0151] Embodiment 56 includes the method of embodiment 55, wherein administering includes inhalation, injection, or intravenous administration.

[0152] Although certain embodiments have been illustrated and described herein, it should be understood that the claims are not limited to the specific forms or arrangements of parts described and shown. Exemplary embodiments are disclosed herein, and although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It should be understood, therefore, that the embodiments may be practiced otherwise than as specifically described.

[0153] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for automated production of adeno-associated virus (AAV) viral vectors, comprising: (a) introducing an artificial mammalian AAV virus producer cell into a completely closed cell engineering system, wherein the artificial mammalian AAV virus producer cell is integrated into the genome; i. adenoviral helper genes including the E2A and E4Orf6 genes under the control of a first derepressed promoter; ii. AAV genes including the Rep and Cap genes under the control of a second derepressed promoter; iii. a virus-associated non-coding RNA under the control of a third derepressed promoter, and iv. introducing the first, second, and third derepressed promoters, (b) transducing said mammalian AAV viral producer cell with a vector encoding a gene of interest to produce a transduced viral producer cell; (c) treating said mammalian AAV virus-producing cells with a binding partner of said inhibitory element; (d) activating the first, second, and third derepressed promoters; and (e) growing the transduced virus-producing cells and producing the AAV viral vector in the transduced virus-producing cells; (f) isolating the viral vector, A method, wherein (a)-(f) are performed in a closed, automated process.

2. 2. The method of claim 1, wherein the artificial mammalian AAV virus producer cell is a mammalian cell culture.

3. The method of claim 2 , wherein the mammalian cell culture is a suspension culture.

4. The method of any one of claims 1 to 3, wherein the artificial mammalian AAV virus producer cell is a Chinese Hamster Ovary (CHO) cell.

5. The method of any one of claims 1 to 3, wherein the artificial mammalian AAV virus producer cell is a human cell.

6. 6. The method of claim 5, wherein the human cell is a human embryonic kidney (HEK) cell.

7. Each of the derepressed promoters contains a functional promoter and two tetracycline operator sequences (TetO ２ 7. The method of claim 1, wherein the repression element is a tetracycline repressor protein.

8. 8. The method of claim 7, wherein said treating comprises treating with doxycycline.

9. The method according to any one of claims 1 to 8, wherein the target gene is a gene of therapeutic interest.

10. The amount of AAV viral vector produced is at least about 10 １０ The method according to any one of claims 1 to 9, wherein the vector is a viral vector.

11. The closed automated process comprises: (a) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor; (b) automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

12. The method of any one of claims 1 to 11, wherein the transducing comprises viral infection, electroporation, liposomal transfection, or membrane disruption.

13. 1. A method for automated production of adeno-associated virus (AAV) viral vectors, comprising: (a) a mammalian cell that stably expresses one or more nucleic acids encoding TetR and / or TetR-KRAB; i. a first nucleic acid encoding adenoviral helper genes including the E2A gene, the E4Orf gene, and a virus-associated non-coding RNA under the control of a first derepressed promoter; ii. a second nucleic acid encoding AAV genes including the Rep and Cap genes under the control of a second derepressed promoter; and iii. Optionally, transducing with a third nucleic acid encoding a gene of interest under the control of a third derepressed promoter; (b) treating the mammalian cell with a binding partner of TetR; (c) activating the first, second, and third derepressed promoters; and (d) expanding the transduced cells and producing the AAV viral vector within the transduced cells; (e) isolating the viral vector, A method, wherein (a)-(e) are performed in a closed, automated process.

14. The method of claim 13 , wherein the mammalian cells are a mammalian cell culture.

15. 15. The method of claim 14, wherein the mammalian cell culture is a suspension culture.

16. The method according to any one of claims 12 to 15, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

17. The method according to any one of claims 12 to 15, wherein the mammalian cell is a human cell.

18. 18. The method of claim 17, wherein the human cell is a human embryonic kidney (HEK) cell.

19. The method of any one of claims 12 to 18, wherein said treating comprises treating with doxycycline.

20. The method according to any one of claims 12 to 19, wherein the target gene is a gene of therapeutic interest.

21. The amount of AAV viral vector produced is at least about 10 １０ The method according to any one of claims 12 to 20, wherein the vector is a viral vector.

22. The closed automated process comprises: (a) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor; (b) automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

23. The method of any one of claims 12 to 22, wherein the transducing comprises viral infection, electroporation, liposomal transfection, or membrane disruption.

24. A method for automated production of lentiviral vectors, comprising: (a) introducing an artificial mammalian lentiviral vector producing cell into a completely enclosed cell engineering system, wherein the artificial mammalian lentiviral vector producing cell is integrated into the genome; i. a regulator of lentiviral virion protein expression (REV) gene under the control of a first promoter; ii. a lentiviral envelope gene under the control of a second promoter, and iii. a lentivirus group specific antigen (GAG) gene and a lentivirus polymerase (POL) gene, both under the control of a third promoter; introducing a nucleic acid sequence flanked at both the 5' and 3' ends by sequences resulting from recombination of transposon-specific inverted terminal repeats (ITRs); (b) transducing said mammalian lentiviral vector producing cells with a vector encoding a gene of interest to produce a transduced viral producing cell; (c) activating the first, second, and third promoters; and (d) growing the transduced virus-producing cells and producing the lentiviral vector in the transduced virus-producing cells; (e) isolating the viral vector, A method, wherein (a)-(e) are performed in a closed, automated process.

25. 25. The method of claim 24, wherein the artificial mammalian lentiviral vector producing cell is a mammalian cell culture.

26. 26. The method of claim 25, wherein the mammalian cell culture is a suspension culture.

27. The method of any one of claims 24 to 26, wherein the artificial mammalian lentiviral vector producing cell is a human cell.

28. 28. The method of claim 27, wherein the human cell is a human embryonic kidney (HEK) cell.

29. The method according to any one of claims 24 to 28, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

30. The method according to any one of claims 24 to 29, wherein the lentiviral envelope gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene.

31. The method of any one of claims 24 to 30, wherein the first promoter, the second promoter and the third promoter are derepressed promoters.

32. 32. The method of claim 31, wherein the artificial mammalian lentiviral vector producing cell further comprises integrating repression elements of the first, second, and third derepressed promoters into its genome.

33. 33. The method of claim 32, wherein each of the derepressed promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repression element is a tetracycline repressor protein.

34. 34. The method of claim 33, further comprising a Krüppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

35. 35. The method of any one of claims 24 to 34, wherein the transposon-specific ITRs are lepidopteran transposon (PIGGYBAC®) ITRs.

36. The method according to any one of claims 24 to 35, wherein the target gene is a gene of therapeutic interest.

37. The amount of lentiviral vector produced is at least about 10 １０ The method according to any one of claims 24 to 36, wherein the vector is a viral vector.

38. The closed automated process comprises: (a) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor; (b) automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

39. The method of any one of claims 24 to 38, wherein the transducing comprises viral infection, electroporation, liposomal transfection, or membrane disruption.

40. A method for automated production of lentiviral vectors, comprising: (a) introducing mammalian cells into a completely enclosed cell engineering system; (b) a mammalian cell; i. a first nucleic acid encoding a regulator of lentiviral virion protein expression (REV) gene under the control of a first promoter and an envelope glycoprotein gene under the control of a second promoter; ii. a second nucleic acid encoding a gene of interest under the control of a third promoter, and iii. Transducing with a third nucleic acid encoding both a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene under the control of a fourth promoter; (c) expanding the transduced cells and producing the lentiviral vector in the transduced cells; (d) isolating the viral vector, A method, wherein (a)-(d) are performed in a closed, automated process.

41. 41. The method of claim 40, wherein the mammalian cell is a mammalian cell culture.

42. 42. The method of claim 41 , wherein the mammalian cell culture is a suspension culture.

43. The method of any one of claims 40 to 42, wherein the mammalian cell is a human cell.

44. 44. The method of claim 43, wherein the human cell is a human embryonic kidney (HEK) cell.

45. The method according to any one of claims 40 to 44, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

46. 47. The method of any one of claims 40 to 46, wherein the envelope glycoprotein gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene, and the VSV-G gene and the REV gene are codon-optimized.

47. The method according to any one of claims 40 to 46, wherein the target gene is a gene of therapeutic interest.

48. The amount of lentiviral vector produced is at least about 10 １０ The method according to any one of claims 40 to 47, wherein the vector is a viral vector.

49. The closed automated process comprises: (a) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor; (b) automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

50. 50. The method of any one of claims 40 to 49, wherein the transducing comprises viral infection, electroporation, liposomal transfection, or membrane disruption.

51. An AAV viral vector produced by the method according to any one of claims 1 to 23.

52. A lentiviral vector produced by the method of any one of claims 14 to 50.

53. A method of treatment with an AAV vector, comprising: A method comprising administering to a mammalian subject the AAV vector of claim 51.

54. 54. The method of claim 53, wherein said administering comprises inhalation, injection, or intravenous administration.

55. A method of treatment with a lentiviral vector, comprising: A method comprising: a. administering to a mammalian subject the lentiviral vector of claim 52.

56. 56. The method of claim 55, wherein said administering comprises inhalation, injection, or intravenous administration.

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