Automated production of viral vectors

Abstract

To provide an automated method of producing viral vectors, utilizing engineered viral vector-producing cell lines, or packaging cells, within a fully-enclosed cell engineering system.SOLUTION: A method for automated production of a viral vector, includes: introducing an engineered viral producer cell into a high-temperature chamber of a fully enclosed cell engineering system; transducing the engineered viral producer cell with a vector encoding a gene of interest to produce a transduced viral producer cell; expanding the transduced viral producer cell and producing the viral vector within the cell; transferring the expanded producer cell to a downstream processing module; and isolating the viral vector and purifying the viral vector, the above-described steps being performed in a closed and automated process. Exemplary viral vectors that can be produced include lentivirus vectors, adeno-associated virus vectors, baculovirus vectors and retrovirus vectors.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure provides a method for automated production of viral vectors that utilizes engineered viral vector-producing cell lines, or packaging cells, within 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.

[0002] [Background technology] Viral vectors are extremely important 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 largely expensive, time-consuming, and cumbersome. Furthermore, methods that rely on crosslinking platforms (e.g., AAV) or multiple transient transfections (e.g., lentivirus) may result in vector yields that are too low or require too much plasmid DNA to support most therapeutic applications. Additionally, for small-scale viral vector production, large batch processes may not be necessary or desirable.

[0003] The benefits of automating viral vector production include labor-hour savings associated with the use of automation, improved product consistency, reduced room cleanliness, reduced cleanroom footprint, reduced training complexity, and improved scale and logistics tracking. Additionally, software can be used to streamline documentation processes by using automatically generated electronic batch records to provide a history of all processing equipment, reagents, operator identification, in-process sensor data, and more.

[0004] Automated, self-contained systems for optimal production of viral vectors in engineered mammalian cells could revolutionize the field of gene therapy. There is an urgent need for technologies that allow for controlled viral production for large-scale or small-scale mass production, resulting in 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 viral vectors, comprising: introducing artificial viral producer cells into a fully enclosed cell engineering system; transducing the artificial viral producer cells with a vector encoding a gene of interest to produce transduced viral producer cells; expanding the transduced viral producer cells to produce the viral vector in the transduced viral producer cells; transporting the expanded producer cells to a downstream processing module; isolating the viral vector; and purifying the viral vector, wherein steps (a) through (e) are performed in a closed, automated process.

[0006] Also provided herein is a method for automated production of viral vectors, comprising: introducing packaging cells into a fully enclosed cell engineering system; transducing the packaging cells with one or more vectors encoding viral helper genes, viral packaging genes, and a gene of interest to produce transduced cells; growing the transduced cells and producing viral vectors in the transduced cells; transporting the grown cells to a downstream processing module; isolating the viral vectors; and purifying the viral vectors, wherein steps (a) through (e) are performed in a closed, automated process. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows a flow diagram for automated production of viral vectors according to embodiments herein. [Figure 2]FIG. 2 illustrates a closed automated cell engineering system as described in embodiments herein. [Figure 3] FIG. 3 illustrates a laboratory space containing an exemplary closed automated cell engineering system as described in embodiments herein. [Figure 4] FIG. 4 shows a diagram of a viral vector production process that can be carried out in a cassette in a closed, automated system, as described in embodiments herein. [Figure 5] FIG. 5 shows a flow diagram of the process within the automated cell engineering system described herein. [Figure 6] FIG. 6 shows a block diagram of downstream processing according to an embodiment herein. [Figure 7] FIG. 7 shows a flow diagram of downstream processing according to an embodiment herein. [Figure 8] 8A-8D show components of a downstream processing module according to embodiments herein. [Figure 9A] FIG. 9 illustrates an exemplary software control design for use with a downstream processing module according to embodiments herein. [Figure 9B] FIG. 9 illustrates an exemplary software control design for use with a downstream processing module according to embodiments herein. [Figure 10] 10A and 10B show two views of a downstream processing module according to an embodiment of the present invention.

[0008] [Mode for Carrying Out the Invention] The use of the word "a" or "an," when used in conjunction with the word "comprising" in the claims and / or specification, may mean "one," but may also be consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0009] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being used to determine the value. Typically, this term means including 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.

[0010] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0011] As used in the specification and claims, the terms "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "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. Furthermore, the compositions, systems, cells, and / or nucleic acids of the invention can be used to achieve any of the methods described herein.

[0012] In embodiments, provided herein are methods for automated production of viral vectors. The automated methods described herein are suitably performed in a closed, automated process.

[0013] A "viral vector" produced by the methods described herein preferably refers to a product virus that can be used to introduce nucleic acid molecules into cells in vitro, in vivo, or ex vivo for therapeutic or industrial purposes. Viral vectors produced by the various methods described herein can be harvested or isolated and stored until their ultimate desired application.

[0014] FIG. 1 shows a block diagram of the automated production process flow described herein.

[0015] Preferably, the methods described herein involve introducing artificial virus-producing cells into a fully enclosed cell engineering system. As referred to herein, an "artificial virus-producing cell" utilized in the 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.

[0016] As referred to herein, the term "introducing" may refer to 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.

[0017] In embodiments, the methods described herein are configured to perform multiple rounds of one or more of feeding, washing, and monitoring artificial virus-producing cells. These various actions may be performed in any order, alone, or in combination with other actions. In embodiments, concentrating the cells includes centrifugation, removal of supernatant after sedimentation, or filtration. Preferably, the optimization process further includes adjusting centrifugation or filtration parameters in a self-regulating process.

[0018] The methods described herein preferably further comprise transducing the artificial virus-producing cells with a vector encoding a gene of interest to produce a transduced virus-producing cell.

[0019] 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 the exogenous nucleic acid molecule within the cell, inducing a phenotypic change in the cell. The transduced nucleic acid molecule may be integrated into the host cell's genomic DNA 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 the use of transfection systems such as liposome-, lipid-, or polymer-based systems, and can also include the use of mechanical transfection such as a gene gun, electroporation, and the like.

[0020] 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 can 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 desired host cells by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection. Vectors may include various regulatory elements, including a promoter.

[0021] "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 sequences) and after (3' non-coding sequences) the coding sequence. In some embodiments, the gene is integrated in multiple copies. In some embodiments, the gene is integrated in a predetermined copy number.

[0022] 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", when referring to a nucleic acid sequence such as a coding sequence or control sequence, refers to a nucleic acid sequence, e.g., a gene, that is not normally linked 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 codons different from the native gene). Allelic variations or naturally occurring mutational events do not give rise to heterologous DNA as used herein.

[0023] Preferably, the gene of interest transduced into the virus-producing cells is a therapeutic gene. As used herein, "therapeutic gene" refers to any functionally relevant nucleotide sequence. Thus, therapeutic genes of the present disclosure can include any desired gene encoding a protein missing or missing from the target cell genome or encoding a non-native protein with a desired biological or therapeutic effect (e.g., antiviral function), or the sequence can correspond to a molecule with antisense or ribozyme function. Representative (non-limiting) examples of suitable therapeutic genes include those used to treat inflammatory, autoimmune, chronic, and infectious diseases, including disorders such as AIDS, cancer, neurological disorders, cardiovascular diseases, and hypercholesterolemia; various blood disorders, including various anemias, thalassemias, and hemophilias; and genetic defects such as cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, and emphysema. Several antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of mRNA) useful for antisense therapy of cancer and viral diseases have been described in the art and are examples of suitable target genes for treatment.

[0024] In exemplary embodiments, the methods described herein, and thus the therapeutic gene, are useful for producing viral vectors for ultra-rare disease applications. While such diseases may not require large amounts of viral vectors (such as when one or a few patients, or 10 patients, or 100 patients require treatment), sterility, reproducibility, and process control are crucial in such applications. The methods described herein, which utilize a closed, automated system, allow for the desired level of control of production.

[0025] In embodiments, the method further includes growing the transduced virus-producing cells and producing viral vectors in the transduced virus-producing cells. As described herein, the method of growing the transduced virus-producing cells preferably includes at least one 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 for the production of a suitable or desired number of viral vectors. In some embodiments, the number of virus-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 an individual cell.

[0026] As shown in Figure 1, transduction 104 and expansion 106 are suitably carried out within fully enclosed cell engineering systems 102. Preferably, these fully enclosed cell engineering systems are automated systems.

[0027] As described herein, a "fully closed cell engineering system" refers to a closed system preferably including multiple chambers, with each of the steps of the various methods described herein occurring 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 suitable for cell growth (e.g., approximately 37°C), with at least one of the multiple chambers maintained at a refrigerated temperature (e.g., approximately 4-8°C). "Fully closed" preferably refers to multiple chambers interconnected, including via various tubing or other fluid connection pathways and connections, to maintain the cleanliness and suitable sterility of the fully closed system.

[0028] As described herein, in embodiments, the provided methods 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 described methods 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 102 useful in the methods described herein is shown in FIG. 2. FIG. 3 illustrates a laboratory space containing an exemplary fully enclosed cell engineering system 102 useful for producing viral vectors in a high-throughput configuration, as described in embodiments herein. In embodiments, each of the closed, automated systems is capable of producing a separate, unique viral vector.

[0029] In embodiments, the transduction and expansion described herein are performed within cassette 202 of a fully enclosed cell engineering system 102 (see FIGS. 2 and 4). Cassette 202 may include a low-temperature chamber for storage of cell culture media, a high-temperature chamber for performing processes involved in viral vector production, the high-temperature chamber being separated from the low-temperature chamber by a thermal barrier and containing 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, waste removal, and uniform gas exchange and nutrient distribution without disturbing the cells in the cell culture chamber. FIG. 4 shows a flow diagram of elements of the viral vector production process that may be performed in cassette 202, as described in embodiments herein.

[0030] FIG. 5 shows a flow diagram of the various components of cassette 202. It depicts a schematic diagram illustrating the connections between cell culture chamber 510 and satellite volume 530. Also depicted in FIG. 5 are various sensors (e.g., pH sensor 550, dissolved oxygen sensor 551), sampling / sample port 552, and various valves (control valve 553, bypass check valve 554), as well as the positioning of one or more fluid pathways 540, which preferably comprise silicone-based tubing components connecting the components. As described herein, the use of silicone-based tubing components allows for oxygen delivery through the tubing components, facilitating gas transfer and optimal oxygenation for cell culture. Also depicted in FIG. 5 is the use of one or more hydrophobic or hydrophilic filters 555 or 556 in the flow paths of the cassette, along with pump tubing 557 and bag / valve module 558. FIG. 5 also shows exemplary locations of an input 580 where artificial virus producer cells (or packaging cells) can be introduced into the cassette 202, as well as an output 590 where the expanded producer cell line (or expanded packaging cells) can be collected and transported to downstream processing module 108.

[0031] As shown in FIG. 1 , after expansion, the expanded production cell line (or expanded packing cell line) is transferred to a downstream processing module 108. As used herein, "transferred" preferably refers to a direct connection between the fully closed cell engineering system 102 and the downstream processing module 108, for example, by connecting the output 590 of the system 102 to the input of the downstream processing module 108 to maintain a closed system and process. As described herein, all elements of the automated production method (from transduction, expansion, isolation to purification) 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) with a direct connection to the downstream processing module 108 to maintain a sterile process. An "automated" process, or "automation" of a process, refers to the control of one or more processes described herein, including a microprocessor for monitoring and altering parameters based on defined or preset conditions or desired characteristics.

[0032] Preferably, downstream processing module 108 performs processes such as viral vector isolation (or separation) and viral vector purification (or purification). As shown in FIG. 1, downstream processing module 108 is preferably a compact, automated, and easily configured and modified processing module. Downstream processing module 108 preferably includes electronic control and operation, as well as mechanical components and sensing. Suitably, the electronic control and mechanical components are durable components in that they do not need to be easily replaced with each virus production process. Downstream processing module 108 also preferably includes disposable cassettes and reagents that are replaceable (and suitably replaced) after each virus production (or at least between different types of viruses produced).

[0033] Figure 6 shows an exemplary block diagram of suitable activities that occur within the downstream processing module 108. As shown, in embodiments, the expanded cell product (sample) is first subjected to a primary recovery to remove the cells from the cell culture medium. The cells are then suitably lysed to expose the product viral vector, preferably by mechanical means (e.g., beads, shaking, etc.) or chemical means (e.g., lysis buffer, detergent, etc.). The viral vector is then isolated using a capture step. This isolation is preferably a bind / elute column step in which the product (viral vector) remains within the matrix while impurities flow through. Suitable column conditions and media are known in the art. Retronectin- or fibronectin-coated surfaces can also be used. This capture step can be repeated as many times as necessary until the total amount of virus is collected. This isolation can also include the use of various affinity columns, including sedimentation columns, as well as chromatography columns and Sepharose columns, which can include functionalized surfaces. Following the initial capture, the solution is suitably titrated from 5 to approximately 7 μL.

[0034] The viral vector is then purified by a polishing step, such as a membrane polishing step in flow-through mode. In such polishing, impurities are absorbed by the membrane, and the desired product (viral vector) flows through. An exemplary polishing step utilizes a strong ion exchanger, such as SARTOBIND® Q ion exchanger (SARTORIUS®, Göttingen, Germany). This polishing step removes unwanted viruses, DNA, host cell proteins, leached protein A, and endotoxins. Exemplary buffers and conditions for performing the polishing step are known in the art.

[0035] Polishing is preferably followed by a pH titration and holding step, which preferably involves lowering the pH to below pH 6 or pH 5, holding for a desired time, and then titrating the pH to about pH 7. The process further preferably involves concentrating or diafiltering the viral particles to achieve a desired concentration.

[0036] After concentration, the viral vector product can be formulated into a final product. This may include the addition of various excipients (e.g., salts, buffers, osmolality adjusters), as well as different media. The viral vector product is then appropriately kept at a low temperature (e.g., about 4-8°C) until it can be administered to a patient, packaged / shipped, or stored.

[0037] In-line 0.2 micron filters are preferably used between the various elements of the downstream processing module to protect against bacterial contamination and remove sediments.

[0038] 7 shows an exemplary flow diagram and exemplary components of the downstream processing module 108 described herein. As shown, the downstream processing module suitably includes various buffers and reagents, as well as columns, that can be changed between runs or for different viral vector systems, as well as components that are not changed, such as pumps, valves, control systems, etc.

[0039] Figure 8A shows an exemplary downstream processing module 108. Figures 8B-8D show disposable / replaceable elements that include different buffers and columns that can be switched.

[0040] 9 shows an exemplary computer control setup for the downstream processing module 108, showing interfaces for controlling the various valves, pumps, etc., as well as monitoring of the system. Also shown are simulated outputs showing various elements that can be monitored, such as pH, conductivity, UV, temperature, pressure, etc.

[0041] 10A and 10B, downstream processing module 108 can include components such as a radio frequency identification (RFID) reader for correlating a user or a particular sample with a particular module. Also shown are pressure sensors, conductivity sensors, pH sensors, UV sensors, peristaltic pumps, and servo valves.

[0042] As illustrated in FIG. 1, following downstream processing module 108, the viral vector product can be passed on to further analysis 110, including determination of viral titer, activity level, contamination, etc.

[0043] 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, through various computer programs and conditions, can determine 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 works together in a coordinated manner at various times and places within the system to achieve optimization. For example, the closed, automated process can 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.

[0044] The automated process can also be based on the intrinsic characteristics of the starting cell population, including, for example, total cell number, cell source, cell density, cell age, 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 to optimize the method, such as lactose, oxygen and carbon dioxide concentrations, flow rates, incubation times, pH, etc. Alternatively, cellular process monitoring can allow for automated characterization of the progression of cell culture sequences from the starting population, allowing for case-by-case adjustment of conditions for optimized final cell culture characteristics.

[0045] In further embodiments, the cell engineering system recycles nutrients, waste products, released cytokines, and / or dissolved gases during various process steps. This recirculation aids in the production of the desired viral vector. Another mechanism for optimizing viral vector production is to modify and control 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 exit the cell culture as required.

[0046] In additional embodiments, the methods and systems described herein can also be used with transient transfection systems. In such embodiments, a method for automated production of viral vectors includes introducing packaging cells into a fully enclosed cell engineering system, transducing the packaging cells with one or more vectors encoding viral helper genes, viral packaging genes, and a gene of interest to produce transduced cells, growing the transduced cells and producing the viral vector in the transduced cells, transporting the grown cells to a downstream processing module, isolating the viral vector, and purifying the viral vector, wherein the elements of the method are performed in a closed, automated process.

[0047] In embodiments utilizing transient transfection, packaging cells can be utilized. As used herein, "packaging cells" refers to cells that do not incorporate one or more viral helper and / or packaging genes into their genome, but instead have these genes added via transfection to produce transiently transfected cells.

[0048] In embodiments, the artificial virus producer or packaging cells utilized in the automated methods are mammalian cells. As used herein, the term "mammalian cells" includes cells derived 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), or CHOK1SV GS-KO (glutamine synthetase knockout) cells, including all variants (e.g., XCEED™, Lonza, Slough, UK). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK293, HEK293T, HeLa cells, or HT1080 cells.

[0049] 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 may be adhesion-dependent. Suspension cultures refer to cells that can be maintained, for example, in culture flasks or large suspension vessels, which 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.

[0050] In embodiments, the methods for producing viral vectors provided herein produce adeno-associated viral (AAV) vectors.

[0051] As used herein, the term "adeno-associated virus (AAV) vector" refers to a small, replication-deficient, non-enveloped virus containing single-stranded DNA of the Parvoviridae and Dependoparvovirus genera. To date, more than 10 adeno-associated virus serotypes have been identified, with serotype AAV2 being the most well-characterized. Other non-limiting examples of AAV serotypes include 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 one serotype and the genome of a second serotype (for example, pseudotype AAV2 / 5 corresponds to an AAV with the genome of serotype AAV2 and the capsid of AAV5).

[0052] As used herein, the term "adenovirus" refers to a non-enveloped virus of the Adenoviridae family, which has an icosahedral nucleocapsid containing double-stranded DNA. 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," The Adenoviruses, Ginsberg, ed., Plenum Press, New York, NY, pp. 497-562 (1984); Strauss, "Adenovirus Infections in Humans," 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: mastadenovirus and aviadenovirus. 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, AV2 and AV5, have been extensively studied and provide most of the general information about adenoviruses.

[0053] In embodiments, the methods for producing viral vectors provided herein produce lentiviral vectors.

[0054] As used herein, the term "lentiviral vector" refers to a small, spherical enveloped virus that contains two single-stranded RNA molecules and belongs to the retrovirus family.Lentivirus contains gag, pol and env genes, and further distinguishes itself from other members of the retrovirus family by having two regulatory genes, namely tat and rev.Lentiviral vectors are 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.

[0055] In embodiments, the methods for producing viral vectors provided herein produce retroviral vectors.

[0056] As used herein, the term " retrovirus " refers to one or more members of the retrovirus family, which is a small, spherical enveloped virus that contains two single-stranded RNA molecules.Retrovirus converts these RNA molecules into DNA, and then integrates 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.

[0057] In embodiments, the methods for producing viral vectors provided herein produce baculoviral vectors.

[0058] As used herein, the term "baculovirus" refers to a rod-shaped virus containing circular dsDNA and one or more members of the Baculoviridae family. It is known to infect and replicate primarily within insect larvae. The baculovirus expression vector system is well established and highly useful for producing proteins in eukaryotic cells (Summers et al., 2006).

[0059] In additional embodiments, the method preferably utilizes insect cells as virus-producing 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, used for the expression and manufacture of proteins and / or baculovirus vector production.

[0060] In embodiments, the present methods suitably utilize Sf9 cells. As referred to herein, "Sf9 cells" are an insect cell line derived from the pupal ovary tissue of the worm Spodoptera frugiperda that are commonly used for the expression and manufacturing of proteins and / or baculoviral vector production.

[0061] 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.

[0062] In embodiments, the methods described herein are for producing adeno-associated virus (AAV) viral vectors. Such processes preferably involve introducing an artificial mammalian AAV viral product into a fully enclosed cell engineering system. As used herein, "viral producer cells" refer to cells that contain, or contain more viral helper or viral packaging genes integrated into their genome. AAV viral producer cells preferably contain adenoviral helper genes, including E2A and E4Orf6 genes, integrated into their genome under the control of a first derepressible promoter. Exemplary artificial mammalian AAV viral producer cells suitable for use in the methods for producing AAV viral vectors are described in detail in U.S. Provisional Patent Applications Nos. 62 / 783,589 and 62 / 866,092, which are incorporated herein by reference in their entireties.

[0063] As described herein, the mammalian AAV virus-producing cells utilized in the present methods preferably contain nucleic acid molecules encoding viral helper genes. Viral helper genes include various adenovirus, herpesvirus, and bocavirus genes (see, e.g., 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 helper genes are adenovirus helper genes. As referred to herein, the term "adenovirus helper genes" or "AV helper genes" refers to genes composed of one or more nucleic acid sequences derived from one or more adenovirus subtypes or serotypes that contribute to the replication and packaging of adeno-associated viruses. In some embodiments, the adenovirus helper genes are E1A, E1B, E2A, E4 (including E4Orf6), VA, or a combination thereof, or any other adenovirus helper genes. In an exemplary embodiment, the adenovirus helper genes include both the E2A gene and the E4Orf6 gene. Preferably, an internal ribosome entry site (IRES) element is included between the E2A gene and the E4Orf6 gene. The IRES element initiates translation of the E4Orf6 gene after the E2A gene in the single expression cassette and provides stability to the construct. Such viral helper genes can also be added to packaging cells by introduction using transient transfection.

[0064] In a further embodiment, the automated production method for AAV viral vectors includes an engineered mammalian virus-producing cell containing AAV genes, including the Rep and Cap genes, under the control of a promoter. These AAV genes can also be transiently transfected into viral packaging cells.

[0065] As referred to herein, the term "Rep" gene refers to the art-recognized AAV genomic region encoding viral replication proteins required together to replicate the viral genome, or functional homologs thereof, e.g., 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 forms of Rep") and Rep52 and Rep40 ("short forms of Rep"), or functional homologs 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 may be modified (e.g., by nucleotide insertion, deletion, or substitution) so long as the present rep gene 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).

[0066] 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 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.

[0067] In a further embodiment, the method suitably comprises propagating the transduced cells to produce the AAV viral vector, and then isolating the viral vector.

[0068] In an embodiment, the method steps are performed in a closed automated process.

[0069] In embodiments, the cells utilized in the production methods, including engineered 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 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 13 It is an AAV viral vector.

[0071] In a further exemplary embodiment, a method for automated lentiviral vector production is disclosed, comprising introducing artificial mammalian lentiviral vector-producing cells into a completely sealed cell engineering system. Packaging cells can also be used to produce lentiviral vectors. Exemplary methods for producing lentiviruses are described in U.S. Provisional Patent Application Nos. 62 / 890,904, filed August 23, 2019, and 62 / 949,848, filed December 18, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0072] As used herein, a "lentiviral vector producing cell" refers to a cell that contains, integrated into its genome, the elements necessary to produce a lentiviral vector. These elements can also be introduced into packaging cells to produce a lentiviral vector.

[0073] In embodiments, the method utilizes a lentiviral vector-producing cell that contains integrated into its genome a lentiviral regulator of virion protein expression (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and both a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene under the control of a third promoter. In preferred embodiments, the nucleic acid sequence is flanked on both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination.

[0074] 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.

[0075] The lentiviral envelope (ENV) gene is preferably the vesicular stomatitis glycoprotein (VSV-G) gene, which encodes a polyprotein precursor that is cleaved by cellular proteases into the surface (SU) envelope glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41.

[0076] GAG encodes a polyprotein translated from unspliced ​​mRNA, which is then cleaved by the viral protease (PR) into matrix protein, capsid, and nucleocapsid proteins. Lentiviral polymerase (POL) is expressed as the GAG-POL polyprotein as a result of ribosomal frameshifting 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.

[0077] In a preferred embodiment, the expression cassette is flanked at both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs).

[0078] Exemplary promoters for use in lentiviral vector production cells are known in the art and include derepressible promoters. Preferably, the expression cassette further encodes repression elements of the first, second, and third derepressible promoters. In embodiments, the derepressible promoters include a functional promoter and a tetracycline operator sequence (TetO), and the repression element is a tetracycline repressor protein, as described herein.

[0079] 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 embodiments, the gene of interest is a therapeutic gene of interest.

[0080] 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.

[0081] In a further embodiment, the method comprises suitably isolating the produced lentiviral vector. Methods for isolating the produced viral vector are described herein.

[0082] In an exemplary embodiment, the method is performed in a closed, automated process.

[0083] 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.

[0084] In embodiments, the automated method for producing lentiviral viral 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 13 lentiviral 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 It is a lentiviral vector.

[0085] In embodiments, the method steps are performed in a closed automated process, preferably including 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.

[0086] Also provided herein are methods for treating mammalian subjects, preferably human subjects, using AAV or lentiviral vectors produced according to the various methods described herein.Preferably, this method is used to treat human subjects with a gene of interest, including a gene of interest for treatment.Administration to human subjects can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.

[0087] Additional Exemplary Embodiments Embodiment 1 is a method for automated production of viral vectors, comprising: introducing artificial viral-producer cells into a fully enclosed cell engineering system; transducing the artificial viral-producer cells with a vector encoding a gene of interest to produce transduced viral-producer cells; growing the transduced viral-producer cells and producing the viral vector in the transduced viral-producer cells; transporting the grown producer cells to a downstream processing module; isolating the viral vector; and purifying the viral vector, wherein the above elements are performed in a closed, automated process. Embodiment 2 includes the method of embodiment 1, wherein the artificial virus-producing cells are mammalian cells. Embodiment 3 includes the method of embodiment 2, wherein the mammalian cells are mammalian cell cultures. Embodiment 4 includes the method of embodiment 3, wherein the mammalian cell culture is a suspension culture. Embodiment 5 includes the method of any one of Embodiments 1 to 4, wherein the viral vector is an adeno-associated viral (AAV) vector. Embodiment 6 includes the method of any one of Embodiments 1 to 4, wherein the viral vector is a lentiviral vector. Embodiment 7 includes the method of any one of Embodiments 1 to 4, wherein the viral vector is a retroviral vector. Embodiment 8 includes the method of any one of Embodiments 1 to 4, wherein the viral vector is a baculovirus vector. Embodiment 9 includes the method of any of embodiments 2-8, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. Embodiment 10 includes the method of any one of embodiments 2 to 8, wherein the mammalian cells are human cells. Embodiment 11 includes the method of embodiment 10, wherein the human cells are human embryonic kidney (HEK) cells. Embodiment 12 includes the method of embodiment 10, wherein the human cells are HEK293T cells. Embodiment 13 includes the method of embodiment 1, wherein the artificially produced cells are insect cells. Embodiment 14 includes the method of embodiment 1, wherein the artificial production cells are Sf9 cells. Embodiment 15 includes the method of embodiment 1, wherein the amount of viral vector produced is at least about 10 viral vectors. Embodiment 16 includes the method of any one of embodiments 1 to 15, 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. Embodiment 17 includes the method of any one of embodiments 1 to 16, wherein transducing includes viral infection, electroporation, liposomal transfection, or membrane disruption. Embodiment 18 includes the method of any of embodiments 1-17, wherein isolating comprises passing the expanded production cells through an elution column. Embodiment 19 includes the method of any of embodiments 1-18, wherein purifying includes membrane polishing. Embodiment 20 includes the method of any one of embodiments 1 to 19, further comprising formulating the viral vector. Embodiment 21 is a method for automated production of viral vectors, comprising: introducing packaging cells into a fully enclosed cell engineering system; transducing the packaging cells with one or more vectors encoding viral helper genes, viral packaging genes, and a gene of interest to produce transduced cells; growing the transduced cells and producing viral vectors in the transduced cells; transporting the grown cells to a downstream processing module; isolating the viral vectors; and purifying the viral vectors, wherein the above elements are performed in a closed, automated process. Embodiment 22 includes the method of embodiment 21, wherein the packaging cell is a mammalian cell. Embodiment 23 includes the method of embodiment 22, wherein the mammalian cells are mammalian cell cultures. Embodiment 24 includes the method of embodiment 23, wherein the mammalian cell culture is a suspension culture. Embodiment 25 includes the method of any one of embodiments 21 to 24, wherein the viral vector is an adeno-associated viral (AAV) vector. Embodiment 26 includes the method of any one of embodiments 21 to 24, wherein the viral vector is a lentiviral vector. Embodiment 27 includes the method of any one of embodiments 21 to 24, wherein the viral vector is a retroviral vector. Embodiment 28 includes the method of any one of embodiments 21 to 24, wherein the viral vector is a baculovirus vector. Embodiment 29 includes the method of any of embodiments 22 to 28, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. Embodiment 30 includes the method of any of embodiments 22 to 28, wherein the mammalian cells are human cells. Embodiment 31 includes the method of embodiment 30, wherein the human cells are human embryonic kidney (HEK) cells. Embodiment 32 includes the method of embodiment 30, wherein the human cells are HEK293T cells. Embodiment 33 includes the method of embodiment 21, wherein the packaging cells are insect cells. Embodiment 34 includes the method of embodiment 21, wherein the packaging cells are Sf9 cells. Embodiment 35 includes the method of embodiment 21, wherein the amount of viral vectors produced is at least about 10 viral vectors. Embodiment 36 includes a method according to any of embodiments 21 to 35, 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. Embodiment 37 includes the method of any of embodiments 21-36, wherein transducing includes viral infection, electroporation, liposomal transfection, or membrane disruption. Embodiment 38 includes the method of any of embodiments 21-37, wherein isolating comprises passing the expanded production cells through an elution column. Embodiment 39 includes the method of any of embodiments 21-38, wherein the purifying comprises membrane polishing. Embodiment 40 includes the method of any of embodiments 21 to 39, further comprising formulating the viral vector.

[0088] Although particular embodiments have been illustrated and described herein, it is to be understood that the claims should not be limited to the specific forms or arrangements of parts described and shown. Although exemplary embodiments are disclosed and specific terms are employed herein, 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 is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0089] 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 viral vectors, comprising: (a) introducing artificial virus-producing cells into a completely closed cell engineering system; (b) transducing the artificial virus-producing cells with a vector encoding a gene of interest to produce transduced virus-producing cells; (c) growing the transduced virus-producing cells and producing the viral vector in the transduced virus-producing cells; (d) transferring the expanded production cells to a downstream processing module; (e) isolating the viral vector; and (f) purifying the viral vector; A method wherein (a) through (e) are performed in a closed, automated process.

2. 10. The method of claim 1, wherein the artificial virus-producing cells are mammalian cells.

3. The method of claim 2 , wherein the mammalian cells are in mammalian cell culture.

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

5. The method according to any one of claims 1 to 4, wherein the viral vector is an adeno-associated viral (AAV) vector.

6. The method according to any one of claims 1 to 4, wherein the viral vector is a lentiviral vector.

7. The method according to any one of claims 1 to 4, wherein the viral vector is a retroviral vector.

8. The method according to any one of claims 1 to 4, wherein the viral vector is a baculovirus vector.

9. The method according to any one of claims 2 to 8, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

10. The method according to any one of claims 2 to 8, wherein the mammalian cells are human cells.

11. 11. The method of claim 10, wherein the human cells are human embryonic kidney (HEK) cells.

12. 11. The method of claim 10, wherein the human cells are HEK293T cells.

13. The method of claim 1 , wherein the artificially produced cells are insect cells.

14. The method of claim 1, wherein the artificially produced cells are Sf9 cells.

15. The amount of viral vector produced is at least about 10 １０ The method of claim 1, wherein the vector is a viral vector.

16. 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.

17. The method of any one of claims 1 to 16, wherein the transducing comprises viral infection, electroporation, liposomal transfection, or membrane disruption.

18. 18. The method of any one of claims 1 to 17, wherein said isolating comprises passing the expanded production cells through an elution column.

19. The method of any one of claims 1 to 18, wherein the purifying comprises membrane polishing.

20. The method of any one of claims 1 to 19, further comprising formulating the viral vector.

21. 1. A method for automated production of viral vectors, comprising: (a) introducing packaging cells into a completely closed cell engineering system; (b) transducing the packaging cells with one or more vectors encoding viral helper genes, viral packaging genes, and a gene of interest to produce transduced cells; (c) growing the transduced cells and producing the viral vector within the transduced cells; (d) transferring the expanded cells to a downstream processing module; (e) isolating the viral vector; and (f) purifying the viral vector; A method wherein (a) through (e) are performed in a closed, automated process.

22. 22. The method of claim 21, wherein the packaging cell is a mammalian cell.

23. 23. The method of claim 22, wherein the mammalian cells are in mammalian cell culture.

24. 24. The method of claim 23, wherein the mammalian cell culture is a suspension culture.

25. The method of any one of claims 21 to 24, wherein the viral vector is an adeno-associated viral (AAV) vector.

26. The method of any one of claims 21 to 24, wherein the viral vector is a lentiviral vector.

27. The method according to any one of claims 21 to 24, wherein the viral vector is a retroviral vector.

28. The method according to any one of claims 21 to 24, wherein the viral vector is a baculovirus vector.

29. The method of any one of claims 22 to 28, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

30. The method of any one of claims 22 to 28, wherein the mammalian cells are human cells.

31. 31. The method of claim 30, wherein the human cells are human embryonic kidney (HEK) cells.

32. 31. The method of claim 30, wherein the human cells are HEK293T cells.

33. 22. The method of claim 21, wherein the packaging cell is an insect cell.

34. 22. The method of claim 21, wherein the packaging cells are Sf9 cells.

35. The amount of viral vector produced is at least about 10 １０ The method of claim 21 , wherein the vector is a viral vector.

36. The closed automated process comprises: (c) 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; (d) automatically adjusting one or more of the temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density.

37. 37. The method of any one of claims 21 to 36, wherein the transducing comprises viral infection, electroporation, liposomal transfection, or membrane disruption.

38. 38. The method of any one of claims 21 to 37, wherein said isolating comprises passing the expanded production cells over an elution column.

39. The method of any one of claims 21 to 38, wherein the purifying comprises membrane polishing.

40. 40. The method of any one of claims 21 to 39, further comprising formulating the viral vector.

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