Cell hybrids as host cells for highly efficient production of gene therapy vectors and viral vaccines

Abstract

The present disclosure provides techniques for adapting host cells to increase the production of viral vectors and particles and improve their quality. Cell hybrids are formed from parental cell lines and are either divided into multiple aliquots for testing or cloned. An aliquot having high production capacity and phenotypic characteristics related to virus production, such as optimal levels of intracellular organelles, etc., is selected and used to establish a production cell line. The production cells can be genetically modified to express a transgene encoding viral elements for producing viral vectors or particles having a therapeutic payload. The hybrid production cells generate more and higher functional titers of viral vectors or particles per cell, thereby reducing the production cost of pharmaceuticals for use in gene therapy and immunization.

Description

Technical Field

[0001] Related Applications This patent disclosure claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 350,863, filed on June 9, 2022. The content of the priority application is hereby incorporated by reference in its entirety for all purposes.

[0002] This disclosure generally relates to the production of pharmaceutical compounds containing viral components. This disclosure also relates to the modification, selection, and genetic engineering of host cells for the high-level production of pharmaceuticals with improved biological and pharmacological properties.

Background Art

[0003] In the past decade, virus vector-based therapies in clinical medicine have become a legitimate option. The FDA has approved 12 therapies using virus vectors across three different types of virus vectors: adeno-associated virus (AAV), lentivirus, and herpes simplex virus. Adenovirus vectors are approved as immunogenic compositions for the treatment of infectious diseases such as COVID-19. Currently, approximately 25 virus vector therapeutics are in the late stages of development, and another 120 are in Phase II trials. The number of virus vectors approved for commercial production will increase rapidly (E. Capra et al., McKinsey and Company, 2022).

[0004] The first gene therapy vectors were typically developed for the treatment of rare diseases. As interest in treating more common conditions has grown, higher yields and lower product costs are being demanded. Over the past few years, large-scale contract development and manufacturing organizations (CDMOs) have invested billions of dollars in virus vector production facilities. While this increased interest is promising, the barriers and challenges of virus vector manufacturing have not been solved by the rapid influx of capital and the development of new technologies.

[0005] Currently, the lack of standardization and low yields are part of the problem. Physical properties and functional requirements vary widely between different vectors. Advanced process optimization is still required for each product. The low recovery rate from the chromatography process means that the yield is typically less than 50 percent (M. May, Biotech. Eng. News, August 2, 2021). In comparison, the production of therapeutic antibodies such as Humira® and Rituxan® and biosimilars is carried out using a standardized platform and typically achieves yields exceeding 90 percent.

[0006] The owners of the technology described in this disclosure previously developed a system that increases the production of monoclonal antibodies in producer cell lines by more than fourfold. U.S. Patent No. 10,329,594. Cultured cells such as CHO cells are fused and hybrid cells are selected for high levels of endoplasmic reticulum or Golgi bodies. Unfortunately, the intracellular machinery required to create viral vectors is quite different. Replication of most viruses occurs in the cytoplasm rather than the ER and needs to be regulated intracellularly to achieve accurate vector assembly. SUMMARY OF THE INVENTION

[0007] This disclosure provides techniques for adapting host cells to maximize the production of viral vectors and particles and improve their quality. Cell hybrids are formed from parental cell lines and are either divided into multiple aliquots for testing or cloned. Aliquots with phenotypic characteristics such as high production capacity for virus production and optimal levels of intracellular organelles are selected and used to establish producer cell lines. Producer cells can be genetically modified to express a transgene encoding viral elements for producing a viral vector or particle having a therapeutic payload. Hybrid producer cells generate more, higher-potency viral vectors or particles per cell, thereby reducing the manufacturing cost of pharmaceuticals for use in gene therapy and immunization.

[0008] Generally, the techniques presented in this disclosure can be used to produce viral vectors or particles. This system involves providing a starter population of cultured cells and forming cell hybrids from the starter population, each comprising two or more cells, and genetically modifying the cell hybrids to express viral elements and a drug payload. The cells are then cultured to produce viral vectors or particles that contain the above elements and encapsulate the above drug payload.

[0009] Throughout this disclosure, the terms "fused cell," "cell hybrid," and "engineered cell" are used interchangeably to refer to cells generated by combining two or more parent cells together to create a single cell having organelles and chromosomes from all parents within the combined cell membrane. A "payload" is a polypeptide, polynucleotide, or any other compound or composition encapsulated within a viral capsid or other macromolecular package. Exemplary payloads are reporter genes for cell screening or therapeutic payloads for medical use. A "viral vector" is a capsid configured to cause the expression of nucleotides contained therein when administered in vivo. A "viral particle" is a capsid configured to deliver a protein or other payload into cells when administered in vivo. A "genetically modified" cell contains an expressible transgene, either within the cell's genome or as a plasmid expressed elsewhere in the cell.

[0010] A method of practicing the technology of the present disclosure is to create a production cell line bank that can be a source for manufacturing capsids containing different payloads. A production cell line for the high-efficiency production of viral vectors or particles is generated by providing a starter population of cultured cells and forming cell hybrids from the starter population in which each cell contains the contents of two or more parental cells. The hybrids are divided into multiple aliquots, which are then sampled for testing. The samples are genetically modified to express one or more transgenes encoding elements of the viral system and reporter means for determining how efficiently the genetically modified cells from each aliquot produce viral components or capsids. The reporter means may be the viral component itself or a drug payload. For screening purposes, it is convenient to use a reporter gene encoding an easily expressible gene product such as a protein that generates a light signal such as green fluorescent protein or luciferin.

[0011] For screening purposes, it is often convenient to use transient transfection means, although stable transfection may be used. Multiple transgenes can be transfected into the cells together or separately. Since different viruses and different viral serotypes can be optimally expressed in cells with different phenotypes, it is often beneficial (but not essential) to use the same virus or serotype for the final screening used to express viral vectors or particles for therapeutic use.

[0012] After transfection, the user characterizes each sample by measuring the production and / or quality of viral vectors or particles containing the reporter gene product for each sample. An aliquot containing cells with desired characteristics is grown in culture to establish a production cell line. Optionally, the user can perform one or more additional cycles of aliquoting, characterizing, and growing to further enrich for cells with the desired phenotype and / or virus production ability. If desired, at any point in the process, the cells can be cloned, either as a means of initially aliquoting the cells or in subsequent steps to stabilize cell characteristics.

[0013] Alternatively or additionally, cell aliquots can be separated or selected according to cell characteristics that have been empirically found to be associated with favorable virus production. This can be done, for example, as part of the process of aliquoting, and the cells are sorted or separated according to the levels of any combination of one or more intracellular organelles, such as mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, or nucleoli, and / or one or more cytoplasmic or intracellular characteristics, such as reactive oxygen species (ROS), cellular redox, or pH. Separation or selection based on phenotype can be performed in conjunction with or simultaneously with the evaluation of virus production, and thus the final selection of cells may be a combination of phenotype and production ability.

[0014] The identification and selection of which aliquot or clone to expand can be based on which aliquot contains cell hybrids that produce more viral capsids per cell, or which aliquot produces capsids filled with a higher proportion of reporter gene, or a combination of the two. Depending on the user's purpose, the techniques of the present disclosure can be implemented by using, as a starter cell population, a single cell line such as CHO cells, mouse myeloma NSO cells, mouse myeloma SP2 / 0 cells, human embryonic kidney 293 (HEK293) cells, baby hamster kidney 21 (BHK-21) cells, VERO cells, PER.C6 cells, and HeLa cells, or their autotypic hybrids, or combinations or hybrids of such cells with cells or primary cells from other cell lines.

[0015] For the industrial-scale production of viral vectors and particles for therapeutic and other purposes, cells from established cell banks are genetically modified to express a transgene encoding one or more viral elements and a transgene that constructs or encodes a desired payload. Transfection can be transient or stable (permanent). The transgenes for the virus and payload can be inserted transiently or stably into the genome of the production cells in various combinations. For example, the components required for virus assembly can be stably transfected into a production cell line to establish a production cell line dedicated to a selected type of virus. The dedicated production strain can then be transiently transfected to contain different payloads depending on the clinical purpose.

[0016] The present disclosure includes production cell lines adapted for the high-efficiency or high-quality production of viral vectors or particles established according to any of the foregoing methods and processes. Optionally, the production cell line is genetically modified with a plasmid encoding one or more viral elements (such as the Rep gene and Cap gene of AAV) and optionally a helper plasmid (e.g., a plasmid encoding adenoviral genes that facilitate AAV packaging), whereby the cell is adapted to produce viral vectors or particles containing the above elements. Some production cells of this technology are characterized as hybrids of two or more parental cells derived from a starter cell population, and the production cells contain an expressible transgene encoding one or more viral elements, whereby the cells are configured to produce viral vectors or particles containing the above elements.

[0017] Such production cells may further contain a transgene that constructs or encodes a payload, whereby the cells are adapted to produce viral vectors or particles encapsulating the above payload. Examples of such payloads include one or more of the following: (1) nucleic acids configured to be expressed in a human subject in vivo when administered to the subject, (2) proteins configured to be delivered to the subject's cells when the particles are administered to the subject, (3) nucleic acids or proteins configured to be transfected into cells ex vivo, and / or (4) the above reporter genes.

[0018] Any of the production cells disclosed herein can produce a specified number of viral vectors or particles per cell, as shown below. Alternatively or additionally, the production cells disclosed herein (compared to cells derived from a starter cell population genetically modified with the same transgene) are characterized as producing more viral vectors or particles, producing viral vectors or particles with a higher functional titer, and / or producing a higher percentage of viral vectors or particles filled with a specific payload, as shown below.

[0019] The present disclosure provides a method for producing a virus vector or particle encapsulating a payload, for example, by culturing cells derived from an established production cell line of the present disclosure. The virus vector or particle produced thereby may be formulated as a medicament under GMP conditions for producing a medicament suitable for administration to humans. The virus vector or particle produced thereby may also be configured for ex vivo treatment in a human subject in need thereof.

[0020] The present disclosure provides a method of treatment by administering the virus vector or particle presented above to a subject. Alternatively, the vectors and particles may be used for ex vivo treatment by contacting them with a plurality of cells (usually autologous or allogenic to the subject) and then administering the cells to the subject.

[0021] Exemplary virus systems that can be used in any aspect of the present disclosure are adeno-associated virus (AAV) vectors or particles and their selected serotypes. In principle, the techniques of the present disclosure can be used to establish production cell lines for any virus system such as adenovirus, alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.

[0022] The virus vector produced according to the present disclosure can be used for the treatment of various human diseases or conditions. The payload may be a polynucleotide encoding a gene product for gene therapy of a human subject in need of gene therapy. The payload may also be a target antigen of a pathogenic microorganism such as SARS-CoV-2 or a nucleic acid encoding the target antigen for inducing an immune response against the pathogenic microorganism in a subject in need of an immune response.

[0023] Various aspects, embodiments, features, and characteristics of the present invention are described in the following sections, the appended drawings, and the accompanying claims.

Brief Description of the Drawings

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[0036] The present disclosure provides improved cell lines for manufacturing pharmaceutical products containing viral elements that significantly reduce commercial production costs. The cell lines are obtained by selecting cells from a mixed population for one or more characteristics that non-specifically support virus or virus production, such as the levels of mitochondria, endoplasmic reticulum, Golgi bodies, and / or other desired phenotypic characteristics compared to other cells in a starter mixture. Particularly effective production cell lines can be obtained by preparing cells for functional selection by creating cell hybrids. An expressible gene containing the element of the intended viral vector or particle may be transfected into the cells before or after one or more cycles of fusion and selection.

[0037] 1. Summary Figure 1 is a general diagram outlining a suitable workflow for implementing the technology of the present disclosure.

[0038] Cells from a selected source (e.g., an established cell line) are fused in multiple cycles to generate a population of hybrids that are heterogeneous in their ability to synthesize viral vectors or particles. To obtain cells from the population that are high producers, the population is divided into multiple separate aliquots or clones. As part of the division, the cells may be screened or separated according to specific phenotypic characteristics that are known or suspected to be beneficial for high-level viral capsid production or filling. Samples from the separated aliquots or clones are individually tested for their ability to produce large quantities or high titers of vectors or particles. The aliquots of cells that are now proven to be high producers are grown and used to establish one or more production cell lines.

[0039] Subsequently, the banked producer cells can serve as a source for the industrial-scale production of one or various therapeutic viral vectors or particles, using in particular the same species and serotype of viral components as those used for screening. The cells selected for industrial production can be transfected in the same manner as those used for screening, except that the reporter gene is replaced with the therapeutic payload. The final transfection can be transient, or an inducible promoter can be used to integrate the viral elements into the genome of the producer cells, after which different payloads can be encapsulated into the same viral system by transient transfection.

[0040] Depending on the choice of viral system and the user's purpose, other workflows may be effective. The various aspects of the disclosed technology can be implemented in any combination and in any order that is effective for producing the viral vector or particle intended by the user.

[0041] 2. Benefits of the Technology Depending on the mode of implementation and application, the aspects of the disclosure described herein can be used to select cell hybrids that produce viral vectors and particles with higher functional titers per volume of culture fluid. This has the following advantages: · Reducing the production cost of viral vectors and particles for clinical use, thereby improving access to such therapeutic agents; · Reducing the need for expansion or construction of new GMP manufacturing facilities in response to an increasing market size; · Providing GMP production in kilograms of the finished protein product using relatively small-scale or fewer bioreactors; · Generating established producer cell lines suitable for high-level expression of different vectors and families of vaccines, if desired; · Reducing the cloning or selection procedures required after integration of the gene to be expressed; · Improving the quality of the product; and · High-quality and small-volume research materials are provided, thereby shortening the time required for the start and completion of clinical trials.

[0042] The technology of the present disclosure can also be used to improve the filling of the produced virus capsids, thereby increasing the effective titer of the preparation. Prior to this discovery, most of the AAV capsids produced by host cells were empty (the industry average is that 10 - 30 percent are filled), which has an adverse effect on the effectiveness and safety of AAV pharmaceuticals and increases the risk of immunogenicity. The following provides a technique for selecting cells with higher contents of mitochondria and reactive oxygen species that results in a two-fold increase in the proportion of capsids with payload.

[0043] 3. Basis The technology of the present disclosure for packaging vectors such as lentivirus, adenovirus, and adeno-associated virus (AAV) utilizes the dependence of the cellular compartments of the major proteins involved in virus packaging and replication.

[0044] The physical associations between the endoplasmic reticulum (ER), mitochondria, peroxisomes, and nucleosomes are thought to be involved in the production of viral proteins. The MAM, a transient contact site between the ER and mitochondria, provides a calcium microdomain for cell signaling such as the activation of calcium-dependent metabolic enzymes. Evidence for transport from the ER to mitochondria is associated with the human cytomegalovirus UL37 protein, where the highly conserved sequence promotes the translocation of these proteins to the ER. ++

[0045] Viral mitochondrial apoptosis inhibitor (vMIA) functions in both the ER and mitochondria. In addition to viral proteins, viral RNA can also target mitochondria (Reeves et al., 2007). Other major viruses for which ER and / or mitochondrial transport is important include hepatitis C virus (HCV) and related viruses of the Flaviviridae family, polyomavirus, rotavirus, coronavirus, poliovirus, enterovirus, hepatitis E virus, HIV-1, adeno-associated virus (AAV; reviewed in Williamson et al., 2012; Ravindran et al., 2016). The adenovirus E1B 19K packaging protein is required for AAV viral packaging and localizes to mitochondria.

[0046] Peroxisomes and nucleoli are important organelles for virus production. Peroxisomes function as hubs for reactive oxygen species (ROS), can be formed de novo from the ER, are hijacked by various types of viruses, where viral proteins are sequestered within these organelles. These include HCV, HCMV, and Kaposi's sarcoma-associated herpesvirus (KSHV). The cell nucleus plays a very important role in virus production. For most viruses, both transport from the cytoplasm to the nucleus and, similarly, retrograde signaling from the nucleus to the cytoplasm are important for virus production. There are numerous examples of nuclear transport of viral proteins.

[0047] Viral proteins have been observed to localize to nucleoli, and the functional importance of nucleoli in viral replication has only recently been elucidated. The importance of nucleoli in viral replication has been most studied in HCMV. In another example, the capsid protein of AAV2 has been shown to be sequestered in nucleoli during viral packaging. In yet another example, the Gag protein of Rous sarcoma virus (RSV) is retained in nucleoli, and this retention is important for efficient packaging.

[0048] The technology of the present disclosure utilizes intracellular organelles (exemplified by the endoplasmic reticulum, mitochondria, peroxisomes, and nucleoli) to further enhance virus production and create super manufacturing cell lines. These benefits are realized by engineered cell lines selected for high ER (including enhanced unfolded protein response), mitochondrial content, peroxisomes, or the properties of single or multiple nucleoli, as described in the present disclosure. By selecting intercellular cells from a mixed cell population for higher levels of intracellular machinery or biochemistry that support increased virus production compared to other hybrid or parental cells in the starting mixture, virus production can be increased.

[0049] At least one of the phenotypic characteristics that are not necessarily specific to the production of a particular virus is selected. This characteristic is not simply the expression level of the protein or substitute of interest. Rather, this is a function that supports the production of a wide range of different viruses. Such characteristics include the relative density of intracellular organelles (especially those involved in virus packaging), and the relative levels or concentrations of various different proteins, such as enzymes that aid in the packaging of HIV, adenovirus, and adeno-associated virus (AAV).

[0050] Further enhancement of virus production is achieved by amplifying stable gene copies of virus genes important for packaging and replication, histone modifiers, enhancers of the protein biosynthesis pathway, and transcriptional amplifiers, and it is potentially possible to create unique manufacturing cell lines that can amplify virus production by tens to hundreds of times as described in the present disclosure compared to conventional host cell lines used in the virus production industry.

[0051] 4. Technical Approach Repeated homotypic fusion of virus-producing cells such as HEK293 cells is thought to result in genome shuffling and whole-chromosome amplification within individual cells. Cells with desirable phenotypes that result in enhanced production capabilities are selected. The fused cell hybrids are excellent at producing viral vectors that result in higher titers, higher capsid filling rates, and / or higher infectivity.

[0052] Figure 9A illustrates a suitable workflow for producing an AAV vector using suspension HEK293 cells as the host. Left: Seeding of cells, transfection, and harvesting of AAV particles from lysed cells. Right: Assessment of product quality: Quantification of capsid, viral titer (or capsid concentration), percentage of full capsids, and functional titer by transduction.

[0053] 5. Demonstration of high-level production of virus from fused cells Polyethylene glycol (PEG) was used as a fusogenic agent to perform cell fusion of HEK293 cells multiple times to form autologous hybrids (multiple cells from one cell line).

[0054] For the packaging and production of adeno-associated virus serotype 2 (AAV2), unmodified and engineered HEK 293F were transfected with a helper plasmid, a viral plasmid containing AAV Rep and Cap proteins, a transfer plasmid expressing NeonGreen fluorescent protein under the control of a constitutive cytomegalovirus (CMV) early promoter adjacent to the AAV2 inverted terminal repeat (ITR), and an additional plasmid expressing microRNA mi342 under the control of a ubiquitous CMV promoter. Transfection was performed using linear polyethyleneimine (PEI). After transfection, the crude virus was extracted from the cell lysate and the virus was recovered by centrifugation. Quantitative PCR was used to measure the copy number of the virus produced by the unmodified and engineered HEK293 cell lines.

[0055] Figure 9B shows the number of viral particles that can be produced from the fused cells. The packaged AAV2 viral genome was measured by quantitative PCR after DNaseI treatment. Primers targeting the NeonGreen gene were used for quantification, and a commercially available AAV2 reference virus with a known vg / ml from Vigene Biosciences was used to determine the vg / ml of the samples. The engineered HEK 293F showed a 2.5-fold increase in the produced viral genome compared to the unengineered HEK 293F cell line.

[0056] 6. Detailed protocol As an example (and without meaning any limitation to the invention described in the claims and its equivalents), a cell line for producing an AAV vector was obtained according to the following protocol:

[0057] Step 1: Production of hybrids. Using a starting cell population of HEK293 cells, cell hybrids were created by using polyethylene glycol as a fusing agent and promoting cell contact in combination with gentle centrifugation. The hybrids were cloned. Each clone was aliquoted and sampled for transfection testing.

[0058] Step 2: Transfection. The sampled hybrid cell clones in the suspension were transfected using a chemical-based method with a lipid polymer that complexes with the negatively charged DNA to form lipoplexes by electrostatic interaction. For transfection, three plasmid vectors were used: 1) a transfer vector that expresses a fluorescent protein under the control of a ubiquitous CMV promoter cassette adjacent to the AAV inverted terminal repeat sequence, 2) a helper vector cassette that expresses the adenovirus E4 gene for AAV DNA replication, the adenovirus E2a gene, and the adenovirus VA RNA (viral associated RNA) gene that enhances the stability of AAV mRNA and promotes AAV capsid transcription, and 3) a packaging vector that expresses the Rep and Cap protein-specific serotypes (AAV1, AAV2, and AAV5) to be assayed. Cells were harvested, lysed, and assayed for AAV production 72 hours after transfection.

[0059] Step 3: Determination of the production ability of the cloned hybrids. The AAV genome copy number was measured by real-time quantitative PCR. The cell lysate was treated with DNaseI to remove non-viral host genomic DNA. Real-time quantitative PCR with fluorescence detection was performed to determine the viral genome copy number. The DNA primers bound to the coding region of the fluorescent reporter in the transfected transfer vector in the assembled AAV, and the copy number was detected using fluorescence (the method used in the previous figure).

[0060] Step 4: Determination of AAV serotype-specific capsids. Biolayer interferometry (BLI) is an opto-biosensing technology that analyzes biomolecular interactions in real time without the need for fluorescent labeling. The amount of AAV virus in an unknown sample was quantified using the interference pattern or phase shift of white light generated by an analyte sample binding to an immobilized ligand on a biosensor probe. Small biosensors that specifically bind to AAV capsid proteins for multiple serotypes (AAV1, AAV2, AAV5). For each serotype (AAV1, AAV2, and AAV5), the concentration of the AAV serotype in an unknown sample was back-calculated using a standard curve of a commercially available AAV reference standard with known concentrations measured by other validated methods.

[0061] Step 5: Measurement of the ratio of full capsids to empty capsids. The ratio of full capsids to empty capsids in an unknown sample can be measured by biolayer interferometry. First, the concentration of the AAV serotype in the unknown sample is measured as described above. For each serotype to be measured, AAV capsids at a normalized concentration are first captured and immobilized on the biosensor. After immobilization, the AAV particles are lysed to release the packaged ssDNA, and the ssDNA is captured and measured using a biosensor probe bound to an SSB protein (which specifically binds to ssDNA). For each serotype (AAV1, AAV2, and AAV5), the ratio of AAV serotype in the unknown sample was back-calculated using a standard curve of a commercially available AAV reference standard together with the known ratio of full capsids to empty capsids measured by other validated methods.

[0062] Step 6: Measurement of infectivity titer. For infectivity titer measurement, undiluted samples of AAV1, AAV2, and AAV5 produced using clonal hybrids were infected at various dilutions and added to a fixed population of uninfected HEK293 cells. The infectivity titer was measured in transduction units (TU) per milliliter (mL). Infectivity was determined by quantifying the percentage of fluorescently positive cells by flow cytometry.

[0063] Step 7: Propagation of clones of high-producing organisms. The original aliquot corresponding to the sample showing high levels of capsid production and functional titer was propagated to establish production cell lines for the transduction and expression of other types of viral vectors and particles.

[0064] Figures 10A, 10B, and 10C show that the productivity of the cells fused, cloned, and sampled according to these figures is higher. Host cells were transiently transfected with (1) a transfer plasmid expressing a fluorescent reporter, (2) a packaging plasmid expressing Rep and Cap proteins specific for AAV1, AAV2, or AAV5, and (3) a helper plasmid. Capsid concentration or titer was measured by biolayer interferometry (BLI) using a biosensor that binds to the AAV1, AAV2, or AAV5 capsid. Cumulative capsid productivity (Figure 10A), cell-specific productivity (VP / cell) (Figure 10B), and percentage of full capsids (Figure 10C) of AAV1, AAV2, AAV5 for HEK293 parent, engineered pool (7A), and clone (#17-2). The engineered clone (17-2) showed increases of 3-fold, 9-fold, and 2.5-fold, respectively, compared to the parental host.

[0065] Figures 11A and 11B show the high functional titers of the cells fused, cloned, and sampled according to this figure. Concentrated packaged AAV1, AAV2, and AAV5 viruses were used to infect HEK293T cells at different multiplicities of infection (MOI), and the percentage of infected cells was determined by gating fluorescently positive cells using flow cytometry. Figure 10A: Average functional titers were calculated at three different virus concentrations (dilution range 1:25 to 1:300). Figure 10B: Cell infectivity measured by the percentage of cells showing GFP fluorescence (AAV transfer vector transgene) at three different multiplicities of infection (MOI) in the range of 500 to 50,000 (depending on the serotype).

[0066] Figure 12 shows the improvement in the percentage of capsids containing the nucleic acid payload achieved in this figure. Host cells were transiently transfected with a transfer plasmid expressing a fluorescent reporter, a packaging plasmid expressing Rep and Cap proteins specific for AAV1 or AAV2, and a helper plasmid. The full / empty ratios of AAV1 and AAV2 in the lysed cells were measured by BLI. The engineered HEK293T pool (4C1) clone (#40) selected for high mitochondria (top 10%) and high reactive oxygen species (top 10%) showed a two-fold increase in the full / empty ratio.

[0067] 7. Preparation of cell hybrids Individual high-producing cells can be selected from any cell population that is heterologous in this regard, as described in the following section. Many single cell lines (such as CHO cells and HEK-293 cells) are inherently diverse enough with respect to gene content and intracellular machinery in a growing cell population that they can be directly screened and selected for high-producing cells from standard culture.

[0068] Optionally, to improve the final product yield or enhance the screening process, the user may prepare cells for screening by using a combination of one or more techniques that enhance the heterogeneity of virus production levels within the cell population or increase the virus production levels of the cell population or a subpopulation thereof as a whole. Suitable techniques are, for example, techniques that shuffle the genome to modify the cell's genome to increase the copy number contributing to the intracellular machinery involved in virus production or assembly. By modifying or shuffling the genome in this way, many genetic variants having one or more of various different properties including virus production levels and growth rates may be generated.

[0069] The technology of the present disclosure is based in part on the disclosure that cells suitable for virus production can achieve higher production levels by fusing with other cells. Without limiting the practice of the invention, it is hypothesized that fusing two cells is partially additive with respect to the components, genetics, or gene regulation of the cells involved in virus production. It is beneficial if the improved characteristics breed true to the traits of the parental generation. Thus, after the cells are fused, the cells are typically subjected to multiple cultures and selection for the phenotypic characteristics of interest. The resulting cells may be aneuploid or retain all or part of the genome of the parental cells encoding cell components involved in virus production.

[0070] Model cells suitable for fusion are cell lines already used in industrial virus production, such as CHO cells, mouse myeloma NS0 cells, mouse myeloma SP2 / 0 cells, rat myeloma YB2 / 0 cells, human embryonic kidney (HEK) 293 cells, HeLa, Per, C6, HT-1080, Huh-7, baby hamster kidney (BHK-21), and Per-CP cells. In the context of the present disclosure, a "cell line" is a population of cells that can grow continuously, extensively, or indefinitely in tissue culture. The starting cell line is typically heterogeneous with respect to one or more phenotypic characteristics related to the amount of gene product derived from the transgene produced by the cells. The production cell lines obtained in accordance with the present disclosure can produce progeny that are heterogeneous, substantially homogeneous, or clonal when cultured.

[0071] Cell fusion is performed by obtaining a cell mixture of cells to be fused (a plurality of cells from one cell line, or two or more cell lines, or a mixture of at least one cell line and at least one primary cell population). The cell mixture is then subjected to an appropriate fusion protocol, for example, by culturing under culture conditions that promote the formation of hybrids, performing electrofusion, combining with a fusion-inducing virus such as Sendai virus, contacting the cells (e.g., by gentle centrifugation), treating with a fusion-inducing agent such as polyethylene glycol (PEG), or using an effective combination thereof.

[0072] For the purposes of the present disclosure, cells produced by fusing two or more cells can be referred to as autotypic hybrids (cells derived from the same cell line that have fused), isotypic hybrids (cells having the same genotype), allotypic hybrids (cells having different genotypes from different individuals of the same species), and xenotypic hybrids (cells of different species). Autotypic hybrids are typically formed using a cell population that is essentially (i.e., at least 99%) composed of cells from a single cell line. Other types of hybrids are typically formed using cell populations from two or more cell lines that potentially have complementary properties. The present disclosure also includes the fusion of one or more cell populations isolated or obtained from a primary source with themselves or with established or cloned cell lines.

[0073] Cells may be fused into hybrids using any suitable technique. For example, cells may be cultured in the presence of a fusion-inducing agent and / or under culture conditions that promote hybrid formation, or optionally in combination with a fusion-inducing agent such as polyethylene glycol (PEG), and forced into contact, for example by gentle centrifugation. Typically, fused cells are obtained by fusing two cells, although it is also possible to fuse three or more cells. It is recognized that the fusion of two different cell populations generates mixed cell products (isotypic, heterotypic, or heterologous hybrids depending on the parental cell lines) and autotypic hybrids. If desired, autotypic or isotypic hybrids can be separated from heterotypic or heterologous hybrids using a fluorescently labeled antibody or a surface-bound antibody that is specific for a ligand expressed by one of the cell lines in the mixture but not by the other.

[0074] Unless otherwise expressly stated, all such combinations are included within the scope of the present invention. It may be beneficial to repeat cell fusion within the hybrid population to further enhance the effect and / or cross with other cell lines to confer additional beneficial properties on the final cell line. Accordingly, the steps of fusion and selection may be repeated two, three, four, or more times.

[0075] 8. Selection of High-Producing Cell Lines and Desirable Phenotypic Characteristics The present disclosure provides various means for identifying and selecting cell hybrids having the ability to generate high-producing cell lines. For testing purposes, cells are transfected with a reporter gene (e.g., a gene encoding a fluorescent substance such as green fluorescent protein) together with a gene encoding a viral capsid. High-producing strains can be selected based on the generated viral capsid and / or the encapsulated promoter gene product.

[0076] Alternatively or additionally, the cell hybrids can be selected for characteristic phenotypes that generally correlate with high levels of proteins and / or viral products. The valuable insight underlying this technique is that by selecting cells from a mixed cell population for the optimal levels of intracellular machinery or biochemistry that support increased virus production, it is possible to increase the production of biological factors compared to other hybrid cells or parental cells in the starting mixture.

[0077] Such phenotypic characteristics include the relative density of intracellular organelles, particularly those involved in the secretion of host cell-derived proteins or viral particles, and the relative levels or concentrations of enzymes that assist in virus completion or assembly. These include mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, and nucleosomes. Such phenotypic characteristics also generally include the cytoplasm or cellular contents, such as reactive oxygen species, redox transport molecules, and pH aspects.

[0078] Depending on the viral system to be optimized, it may be preferable to have either higher or lower levels of any of these phenotypic characteristics, either alone or in combination. As part of the initial aliquoting or cloning step, the cells can be stained with appropriate vital staining dyes and separated into low (at least 10% or ultimately 5% - 25%), medium (intermediate, 30% - 70%), or high (at most 10% or at most 5% - 25%) aliquots for each characteristic on a cell-by-cell basis using a cell sorting device or other means. Hybrid cells corresponding to any or all of these ranges can be recovered, aliquoted, or cloned and then tested for virus production and potency. The ranges determined to be advantageous can then be used as additional criteria for finding other high-producing aliquots or clones for the relevant viral serotype or system.

[0079] The inventors of the present invention have found that it is possible to produce producer cells that generate virus capsids with a two-fold higher proportion of capsids containing an intended pharmaceutical payload, such as a polynucleotide, for the purpose of gene therapy or vaccination, using fused cells selected for higher mitochondrial content and higher reactive oxygen species (ROS) levels.

[0080] 9. Selection for mitochondrial cell content Many viral proteins localize to mitochondria. Mitochondrial content and function are used as criteria for sorting or selecting cells without damaging them using vital staining dyes. Such staining dyes can be obtained commercially, for example, from Invitrogen and Sigma Aldrich. Examples of vital staining dyes for mitochondria include MitoTracker Green FM; MitoTracker Orange CMTMRos; MitoTracker Red CMXRos; MitoTracker Red FM; MitoTracker Deep Red FM; BioTracker 488 Green Mitochondria staining dye; BioTracker 633 Red Mitochondria staining dye; BioTracker 405; and Blue Mitochondria.

[0081] Functional staining dyes for measuring the membrane or redox potential of mitochondria can also be used to sort or select cells with mitochondrially enhanced mitochondrial function. The mitochondrial potential is generated by complexes I, III, and IV and serves as a reliable readout for assessing mitochondrial function. Depolarization of the membrane causes a shift in the fluorescence signal from one wavelength to another. These membrane potential staining dyes are available from Invitrogen and Sigma Aldrich: JC-1 staining dye (Invitrogen T3168, Sigma CS0390), JC-9 staining dye (Invitrogen D-22421), and C10 staining dye (Sigma MAK160, MAK159).

[0082] Additional properties for sorting enhanced mitochondria include mitochondrial calcium, a biosensing dye for measuring superoxide generation, and a dye selective for mitochondria. These include the Rhod-2AM reagent (Invitrogen R1245MP), and MitoSOX Red (Invitrogen M36008).

[0083] Alternatively or additionally, the user can test an expression-based labeling system that introduces a fluorescent protein that targets mitochondria. These are fusion proteins that include a portion that targets the organelle to be labeled or expresses an optical label fused to a protein sequence processed by the organelle. Examples include (Invitrogen: CellLight™ Mitochondria-GFP (C10600); and CellLight™ Mitochondria-RFP (C10505, C10601). Evrogen: pTagCFP-mito (FP117); pTagYFP-mito (FP137); pTagRFP-mito (FP147); pmKate-mito (FP187); pTagGFP2-mito (FP197); pTurboRFP-mito (FP237); pTurboGFP-mito (FP517); pPhi-Yellow-mito (FP607); and pTurboFP602-mito (FP717). Takara Bio: pAcGFP1-Mito Vector (632432); pDsRed2-Mito Vector (632421); pHcRed1-Mito Vector (632434); and pPAmCherry-Mito Vector (632591)).

[0084] After staining with any of these staining dyes, cells having a staining level that is on average at least 1.2-fold, 1.5-fold, 2-fold, or more than 2-fold that of the parental cell line with respect to staining of mitochondria or optically labeled gene products, etc. can be selected (e.g., by flow cytometry and sorting).

[0085] 10. Selection of Peroxisomes, Intracellular Content of Reactive Oxygen Species, and pH Peroxisomes are plastic intracellular organelles found in almost all eukaryotes. They function as external sensors of the cell and as hubs for the β-oxidation of reactive oxygen species (ROS), lipids, and amino acids (reviewed in Smith and Aitchison, 2014). Peroxisomes can be labeled with expression-based labels in which a peroxisome targeting sequence is used to target a fluorescent protein to the peroxisome.

[0086] Examples include: CellLight™ Peroxisome-GFP, BacMam 2.0 (Invitrogen, C10604); pmKate2-peroxi (Evrogen, FP313); and pPhi-Yellow-peroxi-peroxi (Evrogen, FP606).

[0087] After staining with any of these staining dyes, cells having a staining level that is on average at least 1.2-fold, 1.5-fold, 2-fold, or more than 2-fold that of the parental cell line with respect to staining of peroxisomes or optically labeled gene products, etc. can be selected (e.g., by flow cytometry and sorting).

[0088] The present disclosure is the first to show that an increase in the intracellular content of ROS correlates with enhanced viral production. Superoxide anion (O 2-) Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hydroxyl radical (HO·) constitute radical and non-radical oxygen species formed by the partial reduction of oxygen. Intracellular ROS are endogenously generated in the process of mitochondrial oxidative phosphorylation and are associated with various pathological diseases such as cancer, neurodegeneration, and aging.

[0089] Intracellular reactive oxygen species can be measured using fluorescent probes. When oxidized, these reagents emit strong fluorescence and remain localized within the cell. These staining dyes are commercially available and include: Thermo Fisher: CellROX® Green, CellROX® Orange, CellROX® DeepRed, and H2DCFDA. Abcam: DHE (dihydroethidium) assay kit.

[0090] Intracellular redox levels can be measured using OxyBURST Green reagent, RedoxSensor Red CC-1 staining, and reduced calcein, ethidium, fluorescein, Mito Traker probes, and rhodamine. Intracellular pH can be determined using 9-amino-6-chloro-2-methoxyacridine (ACME), BCECF indicator, dextran conjugate, fluorescein and fluorescein derivatives, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), LysoSensor probe, Oregon Green staining dye, pHrodo indicator, SNARF indicator, and thiol-reactive pH indicator.

[0091] 11. Selection of levels of other intracellular organelles Alternatively or additionally, high-producing cells can be identified or selected according to the intracellular content of other intracellular organelles.

[0092] Selection of the endoplasmic reticulum (ER) and / or Golgi apparatus. These organelles play a central role in protein production. Either or both of these can be measured and used as a criterion for sorting or selecting cells without damaging the cells using a vital staining dye, and cells can be selected based on the amount of the relevant staining dye.

[0093] Such staining dyes are commercially available, for example, from Molecular Probes. Examples of vital staining dyes for the ER include ER-Tracker™ Blue-White DPX (E12353); ER Tracker™ Green (glibenclamide BODIPY® FL) (E34251); ER-Tracker™ Red (glibenclamide BODIPY® TR) (34250); DiOC6 (D273); and DiOC5 (D272). Examples of vital staining dyes for the Golgi apparatus include NBD C6-6-ceramide (N1154); NBD C6-sphingomyelin; BODIPY® FL C5-cerimide (D3521); and BODIPY® TR ceramide (D7540).

[0094] Alternatively, or additionally, the user can test an expression-based labeling system that introduces a fluorescent protein that targets the ER or the Golgi apparatus. These are fusion proteins that contain a portion that targets the organelle to be labeled or that expresses an optical label fused to a protein sequence that is processed by the organelle. Examples include: Invitrogen: CellLight™ ER-GFP (C10590); CellLight™ ER-GFP (C10591); CellLight™ Golgi-GFP (C10592); CellLight™ Golgi-GFP (C10593). Evrogen: pmKate2-ER (FP324); pFusionRed-ER (FP420); pTagRFP-Golgi (FP367); pTagRFP-Golgi (FP367); and pFusionRed-Golgi (FP419). From Clontech: pDsRed2-ER Vector (632409); pDsRed-Monomer-Golgi Vector (632480); and pAcGFP1-Golgi Vector (632464).

[0095] After staining with any of these staining dyes, cells can be selected (e.g., by flow cytometry and sorting) that have staining levels that are on average at least 1.2-fold, 1.5-fold, 2-fold, more than 2-fold, or greater than those of the parental cell line with respect to staining of the ER, Golgi apparatus, or optically labeled gene product, etc.

[0096] Selection of nucleoli with high cell content. Nucleoli are the largest nuclear organelles in cells where ribosomal RNA is assembled, transported to the cytoplasm, and supports the protein translation machinery. Nucleoli can be labeled in cells with vital staining dyes. It is also possible to selectively stain this nuclear organelle using a fluorescent probe that targets ribosomal RNA. Examples include: Nucleolar Staining Kit (Abcam, ab139475); NUCLEOLAR-ID Green Detection Kit (Enzo Life Sciences, 51009-500); and SYTO RNASelect Green (Invitrogen, S32703).

[0097] After staining with any of these staining dyes, cells can be selected (e.g., by flow cytometry and sorting) that have a staining level that is on average at least 1.2-fold, 1.5-fold, 2-fold, or more than 2-fold higher than the parental cell line with respect to staining such as nucleoli or optically labeled gene products.

[0098] 12. Characterization of high-producing cell lines Cell hybrids optimized for the production of viral vectors and particles can be characterized by one or more criteria in any combination.

[0099] Suitable criteria include cell karyotype. Chromosome patterns may be characteristic of homotypic and heterotypic cell fusions. The following characteristics may be advantageous for virus growth: · Duplication of chromosome segments · Loss of chromosome segments (less than 90%, 80%, 70%, 60% or 50% of the original segment size) · Differences in heterochromatin distribution and amount (differences of 10%, 20% or more than 20%, and / or differences in heterochromatin distribution of more than 20%) · Translocation events (two or more translocation events on the same or different chromosome segments compared to the parental cell line).

[0100] The producer cells may also be characterized based on cell phenotypes such as intracellular contents such as mitochondria, peroxisomes, reactive oxygen species (ROS), endoplasmic reticulum, Golgi apparatus, nucleosomes, etc. using the above materials.

[0101] 13. Determination of the production capacity and characteristics of producer cells Cell lines or mixed cell populations selected for high-level virus production may be characterized by one or more of several different parameters compared to the parental cell line or the original cell line. For example, the selected cells may have (1) a genome with a higher aneuploidy than the starting cells, including some or all of the genomes of two or more parental cell lines (which may or may not be the same), (2) a higher concentration (e.g., 2 - 5-fold or 4 - 8-fold, or 2-fold, 4-fold, or 8-fold higher) of mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, reactive oxygen species, or other phenotypic characteristics compared to any one or all of the parental cell lines, (3) a significantly higher ability to produce virus per cell or per liter of culture medium (e.g., 2 - 5-fold or 4 - 8-fold, or 2-fold, 4-fold, or 8-fold higher), (4) the ability to produce a specific amount of virus per cell (e.g., more than 50, 65, 75, 100, 150, 200, 300, 500, 2000, 5000, or 20,000 capsids per cell), (5) the ability to produce a specific amount of virus per volume of culture medium (e.g., at least 5, 8, 12, 20, or 30 grams, or 8 - 20 grams or 10 - 50 grams of virus per liter of culture medium), or (6) the ability to produce virus vectors or particles with a higher proportion (50% higher, or 2-fold or 3-fold) of payload-bearing capsids.

[0102] For the purpose of making such comparisons, the production cell line can be compared to a standardized population of the original cell line that is stored in-house as part of the same system or obtained from a reference source. For example, a production cell derived from CHO may be compared to the CRL-12023 cells of the American Type Culture Collection (ATCC®). The present disclosure includes a system for high-level production of virus-based pharmaceuticals that includes both a starting cell line and a production cell line derived from the starting cell line that has a relatively high density of mitochondria and / or reactive oxygen species (ROS) per cell as determined using, for example, one or more of the vital staining dyes listed above.

[0103] 14. Genetic Modification of Production Cells for Synthesizing and Producing Virus Elements To generate a cell line that expresses viral gene products, a production cell or its precursor can be transfected with a single gene encoding virus elements such as proteins and nucleic acids. Often, virus elements are introduced into host cells using multiple vectors.

[0104] Expression of the gene cassette can be placed under the control of the following combinations of mammalian promoters: ubiquitous promoters, endogenous viral promoters (not ubiquitous, e.g., p5 and p19), hybrid promoters, and / or inducible promoters that cause expression of single or multiple gene cassettes in the host cell line. The gene can be arranged in the forward or reverse direction relative to the promoter. The gene or genes may be adjacent to recombination sites (FRT and its variants and / or lox and its variants). Variants of these recombination sites include loxP, lox511, lox2272, FRT, or mFRT71. Site-specific recombinases such as Cre or Flippase are expressed in the same cell to allow site-specific recombination and change the direction of the gene from reverse to forward.

[0105] For example, recombinases can be expressed by transient transfection in which the gene encoding the recombinase is under the control of a ubiquitous or inducible mammalian promoter. In another example, purified recombinase protein or mRNA can be transfected into cells. In another example, recombinases can be delivered using adenovirus, lentivirus, AAV, Moloney murine leukemia virus (MMLV), mouse stem cell virus (MSCV), vesicular stomatitis virus (VSV), or herpes simplex virus (HSV). Multiple genes can be expressed by the same promoter by using polycistronic elements such as T2A, P2A, E2A, F2A, IRES, and IRES2 elements. Inducible promoters cause the expression of a gene or gene cassette upon application of a stimulus that can be chemical (e.g., recombinases such as doxycycline, tetracycline, cumate, Cre or Flippase) or physical (e.g., blue light). The production level of the target protein can be determined during the process of treatment using the transient transfection method for inserting the gene expression cassette.

[0106] Alternatively or subsequently, permanent transfection can be performed to integrate the gene of interest and / or the marker gene into the genome of the cell line. By co-transfecting a transposase and a gene cassette flanked by transposase recognition sites known as transposase inverted terminal repeats, multiple gene integration copies (up to 50 integrated copies per cell) can be achieved.

[0107] Adenovirus, adeno-associated virus (AAV), and lentivirus can be produced by transiently transfecting a cell line with one or more combinations of helper vectors, packaging vectors, envelope vectors, and / or transfer vectors. The gene cassettes of helper vectors, packaging vectors, envelope vectors, and transfer vectors differ depending on the type of virus produced. Helper vectors can express the E2A and E4 genes and the VA RNAs of adenovirus and AAV. In another example, packaging vectors express the Rep and Cap genes of adenovirus and AAV, while different packaging vectors express Gag, Pol, Rev, and their response elements for lentivirus production.

[0108] Alternatively, the Rep gene cassette can be split into two segments (a 5' segment and a 3' segment), and these two segments are joined by a stop cassette containing transcription termination and polyadenylation sequences adjacent to two homologous recombination sites located in cis, such that the Rep or envelope gene can be expressed in an inducible manner. In another preferred example, the E2, E4, and VA cassettes are placed under the control of an inducible promoter in the reverse orientation relative to the promoter. Activation of the EEV is achieved by the delivery of Cre and doxycycline. In a preferred example, loxP and lox511 are used for recombination. Other heterologous recombination sites such as lox2272, FRT, and mFRT71 can be used. If any combination of FRT and mFRT71 recombination sites is used, Flippase also needs to be delivered to the cells. The envelope vector is only required for the production of lentivirus and is not required for the production of adenovirus or AAV. The transfer vector backbone containing the gene of interest is specific to the virus type. The lentivirus transfer vector backbone is composed of 5' and 3' LTRs, the Psi packaging signal, and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The adenovirus and AAV transfer vector backbones contain inverted terminal repeats adjacent to the promoter, the gene of interest, and WPRE.

[0109] Adenovirus or AAV can be produced by transfecting a cell line that does not contain any integrated copies of the helper vector, packaging vector, or transfer vector into a viral vector. In another example, a transfer vector (expressing the gene of interest) is transfected into a cell line containing stable integration of helper elements and packaging elements. In another example, the cell line may have stable integration of the helper vector, packaging vector, and transfer vector.

[0110] Lentiviruses can be produced by transfecting a packaging vector, an envelope vector, and a transfer vector into a parental host cell line. In another example, lentiviruses are produced by transfecting a transfer vector (expressing the gene of interest) into a cell line containing stable integration of a helper element and a packaging element. In another example, virus is produced in a cell line having stable integration of a helper vector, a packaging vector, and a transfer vector.

[0111] Transfection can be performed using liposome-based reagents (e.g., Lipofectamine™ 3000, Expifectamine 293, FuGENE™ HD, X-Fect nanoparticle polymer, Trans-IT Pro reagent, Trans-IT VirusGen, polyethyleneimine), calcium phosphate, electroporation, or infection with an adenovirus, retrovirus, or lentivirus-based vector.

[0112] After transfection, the cells are tested for the production of the intended viral packaging, for example, by enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), or biolayer interferometry (BLI). Cells or clones with increased production of the desired virus are selected. The goal is an increase in virus production that is 1.5, 2, 4, 8, 12, 16, 20, or 100 times higher than that of the parental cell line, and / or production at levels exceeding 10 12 viral genomes / ml or capsids / ml, and / or production exceeding 10 8 infectious units (IFU) per ml of culture under typical manufacturing conditions. The desired virus can also be tested for other desired characteristics such as the ratio of full capsids to empty capsids and the functional titer.

[0113] In principle, transfection can be performed before, during, or after one or more cycles of selection for fusions and other features. For example, fusions and selections can be performed before transfecting a packaging vector, helper vector, and transfer vector containing the gene of interest, thereby establishing a parental cell line suitable for high-level virus production selected by the user. Alternatively, transfection can be performed on the original parental cell line containing the gene of interest, and the production level can be tracked during subsequent fusion and sorting steps, or used to provide another criterion for such sorting. Alternatively, transfection can be performed as an intermediate step, in which case the cells have already been subjected to one or more cycles of selection for fusions and other features such as ER, Golgi apparatus, mitochondria, peroxisomes, nucleosomes, or other proteins (described above in this disclosure). The resulting hybrid is transfected to express the virus of interest and then subjected to further cycles of selection for fusion and expression of the virus of interest and / or other features described above in this disclosure.

[0114] Another option is to develop a cell line that uses a reporter gene as a surrogate for the ultimately produced viral payload, such as a fluorescent viral payload such as secreted alkaline phosphatase, secreted luciferase, red fluorescent viral payload, or green fluorescent viral payload. Again, transfection can be performed, optionally, in one or more cycles using the expression level of the marker as a selection criterion before, during, or after multiple cycles of fusion and selection. This is expected to produce a parental cell line optimized for the expression of the marker viral payload and to retain the beneficial characteristics of the cell line even after further genetic modification to produce a commercially valuable biological product.

[0115] Finally, when a cell line with the expression of the marker virus payload at the desired level is developed, the marker is replaced with the target virus payload. Using the techniques listed above, transfection into the genome can be randomly performed again, and the expression of the reporter gene is suppressed. Alternatively, using a targeted integration technique, the gene related to the reporter gene can be replaced with the gene encoding the target virus payload. Such techniques include, for example, CRISPR / Cas virus payload, CRISPR / Cas-associated transposase (CAST), recombinase cassette exchange (RMCE), zinc finger recombinase (ZFR), or transcription activator-like effector nuclease (TALEN). In this way, the gene of interest is inserted into the genome of the producer cell line or cells derived from the mixture at a position preselected to enable or support a high level of transcription compared to other positions in the genome.

[0116] 15. A method for stably transfecting producer cells using a transposase A transposon is a DNA sequence that can move from one position in DNA via either a (1) copy-and-paste or (2) cut-and-paste mechanism. These have recently emerged as a promising molecular biology toolkit for gene amplification that can introduce up to 50 copies into the genome. Compared to transposons, conventional transfection techniques using chemical-based methods or electric currents can only integrate a single or very few copies of the transgene into the host genome.

[0117] As an example, the class II transposon "Sleeping Beauty" may be used to stably introduce multiple copies of a viral gene into cells. By stably introducing the desired copy number of a specific viral gene into cells as predicted, the benefit of maximizing viral production can be realized. The optimal ratio of viral proteins is important for the accurate packaging of lentiviruses, adenoviruses, and AAVs and can vary depending on the type of virus. The idea of amplifying the copy number of viral genes integrated into cells using class II transposons (Sleeping Beauty and Piggy Bac) can also be applied to class I transposons and CRISPR transposons (CASTs; Mougaikos and Beisel, 2021).

[0118] Multicomponent molecular systems such as the SunTag system can be used to amplify gene expression of viral genes in cell lines without using the Sleeping Beauty transposase system. Global transcription factors such as Tat or p300 are tagged with multiple copies of a SunTag scaffold containing multiple copies of the GCN4 epitope. The cognate scFv fragment is fused to a heterotypic fusion protein consisting of the transactivation domains p53, VP64, p65, and Rta (SSPVP) (see Figure 3). When these components are co-expressed in cells (referred to herein as the supertranscriptional activation complex (STAC)), this can synergistically result in amplified gene expression of viral genes. In contrast to gene copies amplified using a transposase system, such systems can be used to realize the benefit of amplified gene expression in cells containing a small number of integrated gene copies. This allows for amplification beyond the normal levels of the endogenous biological system.

[0119] 16. Viral strains suitable for use in therapy The technology of the present disclosure can be implemented in any viral strain selected by the user with necessary modifications. Table 1 provides some examples of viral gene elements that can be transfected into the production cell lines of the present disclosure for the preparation of lentivirus, adenovirus, and AAV vectors.

Table 1

[0120] 17. Promoters for expressing viral gene elements stably transfected into production cell lines When stably transfecting a production cell line to integrate a viral transgene into the cell's genome, it may be useful for the control of such cells if the promoter used to drive the expression of the viral gene in the production cells is inducible.

[0121] One example is the Cumate-inducible promoter (CymR), a repressor that binds to the Cumate operator sequence (CuO) in the absence of Cumate. In the presence of Cumate, Cumate binds to CymR and enables the activation of genes downstream of CuO. U.S. Patent Nos. 8,728,759 and 7,745,592B2. The tetracycline response element (TRE), which can be induced using doxycycline or tetracycline, is also suitable. Photoinducible promoters such as the blue light-inducible promoter of Gen Target, Inc. can also be used.

[0122] 18. Methods for quantifying viral production levels from cell hybrids Real-time quantitative PCR measures viral transcription and viral genome concentration (vg / ml). Each viral particle typically contains one viral genome. The virus is treated with DnaseI to remove host genomic DNA. Primers that bind to the targeting region in the transfer vector are used, and amplicons are detected either by a probe-based method or by SYBR Green that binds to the amplicon.

[0123] Indirect ELISA and Biolayer Interferometry (BLI) are used to measure total capsid AAV particles. In these measurements, antibodies against capsid proteins abundant in the AAV serotype are utilized. Samples are captured by the capsid antibody and detected using a biotinylated capsid antibody and streptavidin conjugated to HRP for chemiluminescent detection.

[0124] In the case of lentivirus, infectious particles can be measured by indirect ELISA or sandwich ELISA using an antibody against p24. Anti-p24 is used to capture the sample and detected using biotinylated anti-p24 together with streptavidin conjugated to HRP for chemiluminescent detection.

[0125] The viral potency or infectivity titer is the concentration of viral particles capable of transducing cells. The potency can be measured by cell transduction using a fluorescent protein or a chemiluminescent protein as a reporter. The packaged virus is infected or transduced into a cell line at a specific multiplicity of infection (MOI). The proportion of cells expressing the reporter gene is quantified and correlated with the number of viral particles used to transduce the cells.

[0126] 19. Pharmaceutical Payload and Therapeutic Uses Viral vectors and particles produced in accordance with the present disclosure can be used to deliver various pharmaceutical payloads to a human subject in need thereof. Various types of proteins and nucleic acids or combinations thereof are suitable. Treatment is effected by administering to the subject an amount of the vector or particle effective to achieve one or more clinical objectives.

[0127] The techniques of the present disclosure are advantageous for delivering nucleic acids, proteins, or mixtures thereof for the purpose of inducing a specific immune response. Exemplary payloads of immunogenic compositions or vaccines are shown in Table 2. The packaged nucleic acid encodes one or more epitopes from the intended immune target and optionally one or more additional proteins that can act as adjuvants or stimulants to enhance immunogenicity. The target may be an infectious agent such as a pathogenic virus, bacterium, or protozoan. Alternatively, the target may be a cancer cell, in which case the encoded epitope is an epitope expressed by the cancer cell that is specific for the cancer or tissue type.

[0128] For example, the techniques of the present disclosure can be used to prepare a composition that induces a response against the SARS-CoV-2 virus for the purpose of preventing or treating COVID-19. Representative immunogenic epitopes can be obtained from any one or more of the four structural proteins of SARS-CoV-2, namely, the membrane glycoprotein (M), envelope protein (E), nucleocapsid protein (N), and spike protein (S). Most current vaccines against SARS-CoV-2 typically include or encode the entire spike protein. Methods for optimizing the spike protein have recently been discussed in F. Heinz & K. Stiasny, NPJ Vaccines (2021) 6:104. [Table 2]

[0129] The techniques of the present disclosure can also be used for gene therapy purposes, for example, for the delivery of nucleic acids encoding gene products that target deleted or defective genes in the subject to be treated or pathogenic cells, particularly cancer cells, in the subject. The purposes of treatment include, but are not limited to, the expression of therapeutic proteins encoded by the nucleic acids (such as cytokines or anti-cancer agents), the expression of essential proteins that the subject cannot produce by itself, or the delivery of gene editing systems such as CRISPR / Cas9 or guide RNAs. Other possible therapeutic payloads include DNA antisense oligonucleotides, DNA aptamers, microRNAs, small interfering RNAs, ribozymes, RNA decoys, and circular RNAs that specifically increase or decrease the expression of specific endogenous genes or infectious agents in the subject. K.Sridharan et al., Br J Clin Pharmacol. 2016 Sep;82(3):659-672.

[0130] Exemplary payloads for gene therapy are shown in Table 3. In the examples shown, the nucleic acids encode therapeutic antibodies (for passive immunization), anti-cancer agents such as cytokines and chemokines (for cancer treatment), and natural human proteins (to facilitate the synthesis of essential factors that may be deleted in the subject, such as in the case of genetic diseases). Tables 2 and 3 are cited from X.Hou et al., Nat Rev Materials 2021, 10:1-17.

Table 3

[0131] 20. Pharmaceuticals and Commercial Products For the preparation and formulation of pharmaceuticals for use in accordance with the present disclosure, standard techniques described in, for example, the latest edition of Remington: The Science and Practice of Pharmacy can be incorporated. The formulations are typically optimized for systemic administration (intramuscular or subcutaneous administration) or for oral or nasal administration (for example, to stimulate the mucosal immune system).

[0132] Preparations of viral vectors and particles may be provided as one or more unit doses (either combined or separately), each containing an amount of pharmaceutical payload effective for the treatment of a selected disease, infection, or clinical condition. Commercially available products may include a device such as a syringe for administering the agent or composition into or around the target tissue of a subject in need thereof. The product may include or be accompanied by a package insert describing the use of the vector or particle in the treatment of the indications for which it is adapted and approved and the attendant benefits.

Examples

[0133] Example 1: Protein Production by Cell Hybrids In this example, CHO cells were fused and cells with a high endoplasmic reticulum (ER) content were selected for the purpose of maximizing protein production.

[0134] CHO-K1 cells were exposed to a fusion procedure using PEG. The cells were allowed to recover for one week and then the procedure was repeated a total of three times. After recovery from the third fusion, the cells were stained with an ER-tracking vital staining dye (ER-Tracker™ Green (glibenclamide BODIPY® FL), Invitrogen, E34251) and sorted using a FACSAriaII™ cell sorter (BD Biosciences). The 10% viable population showing the highest staining intensity with the ER-Tracker staining dye was collected. After allowing the culture to recover for two weeks, the cells were exposed to a final fusion, stained with the ER-tracking staining dye, and analyzed using an LSRII™ flow cytometer (BD Biosciences).

[0135] To measure protein production in the fused cells and parental CHO population, the cells were transfected to express secreted alkaline phosphatase (SEAP). Transfection was performed as follows: 1. Centrifuge 1.10 6 individual cells. 2. Discard the supernatant. 3. Resuspend in 100 μL of Cell Line Nucleofector™ Solution T. 4. Add 2 μg of SEAP expression plasmid. 5. Transfer to an electroporation cuvette. 6. Electroporate using Amaxa™ Nucleofector II and preset program U-023. 7. Add 0.5 mL of growth medium. 8. Transfer the cells to a 6-well plate containing 1 mL of growth medium per well.

[0136] Figure 7 shows the results (specific productivity of secreted alkaline phosphatase). The expression of the marker protein (SEAP) in the fused cells shows an improvement of more than 4-fold.

[0137] Example 2. Generation of AAV packaging cell lines containing Rep-Cap and EEV gene cassettes using Piggy Bac and Sleeping Beauty Transposase System 2.1 Generation of cell lines containing Rep-Cap (fHEK-RC) A vector containing the Rep-Cap gene cassette (Figure 2A) is transfected into fused HEK 293F (fHEK) cells by electroporation using a Bio-Rad Gene Pulser. Vector DNA and the Piggy Bac transposase vector are added to the cell suspension, followed by electroporation using a single-pulse exponential decay. After transfection, selective medium containing the selection concentration of blasticidin is added to the medium, and viable cells containing stable integration of Rep-Cap are transfected into the EEV cassette using the Sleeping Beauty transposase system (Figure 2B).

[0138] Figure 3A: Rep-Cap inducible integration gene cassette. The Rep-Cap gene cassette is flanked by left and right Piggy Bac transposon inverted terminal repeats (LTR and RTR, respectively) and contains the following: 1) the coding regions of Rep and Cap expressed by the viral endogenous promoter, p5, followed by the SV40 polyA tail. The Rep gene cassette is divided into a 5' region and a 3' region. A stop cassette containing a transcription termination sequence flanked by two loxP sites is located between the 5' Rep gene cassette and the 3' Rep gene cassette in the coding region of Rep78. The CMV promoter is used to drive the expression of the blasticidin resistance gene, followed by a polyA tail.

[0139] Figure 3B: Inducible cassette for activation of E2A, E4, and VA RNA (abbreviated as EEV and Cre recombinase). The inducible cassette is flanked by Sleeping Beauty transposon inverted terminal repeats (abbreviated as left and right LTR and RTR, respectively). EEV is in the reverse orientation and flanked by heterologous LoxP sites. When doxycycline is activated, cis recombination occurs in the correct forward direction in EEV and they are expressed in a dose-dependent manner. Cre recombinase is under the control of doxycycline via the TRE promoter (see below). Dox binds to the reverse tetracycline-controlled transactivator (rtTA) to form the Dox-rtTA complex. The Dox-rtTA complex binds to the tetracycline response element (TRE) to activate the downstream target gene. The inhibitor, reverse tetracycline-controlled transsilencer (rtTS), serves two functions: (1) minimizing the leaky expression of the TRE promoter and (2) enhancing rtTA activity in the presence of doxycycline, which functions as a co-activator. Other abbreviations: Cbh, CMV and chicken beta-actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 polyA tail.

[0140] 2.2 Generation of cell lines (fHEK-AAV) containing Rep-Cap and EEV A vector containing the E2A, E4, and VA gene / RNA cassette (see Figure 2B) and the Sleeping Beauty transposase (SBT) is sequentially transfected into fHEK-RC by electroporation using a Bio-Rad Gene Pulser with a single-pulse exponential decay. The transfected cells are grown in selective medium containing blasticidin and hygromycin, and the surviving cells contain stable integration of the Rep-Cap and EEV cassettes.

[0141] Example 3. Generation of AAV packaging cell lines (fHEK-SAAV) containing Rep-Cap and EEV cassettes A vector containing the EEV and STAC cassettes (Figure 3) is transfected into fHEK-RC by electroporation using a Bio-Rad Gene Pulser with a single-pulse exponential decay. The transfected cells are grown in selective medium containing blasticidin and hygromycin, and the surviving cells stably integrate the Rep-Cap, EEV, and STAC gene cassettes.

[0142] Figure 3C: Inducible STAC cassette for amplified synergistic activation of E2A, E4, and VA RNA (EEV). The inducible cassette is flanked by Piggy Bac transposon inverted terminal repeats (abbreviated LTR and RTR for left and right, respectively). The transcriptional co-activator and histone acetyltransferase (HAT), p300, and the HIV-1 Tat transcriptional activator both bind to multiple GCN4 peptides (shown as black squares). Both fusion proteins are expressed under the control of the CMV promoter. The hybrid scaffold SSPVP consists of an scFv fragment that binds to the GCN4 peptide, and the transactivation domains of p53, VP64, and p65 to recruit additional transcription factors, HAT, and co-activators to the basal transcriptional machinery at the promoter.

[0143] An inducible cassette (abbreviated as EEV and Cre recombinase) for the activation of E2A, E4, and VA RNA. The EEVs are in the reverse orientation and are adjacent to heterologous LoxP sites. When doxycycline is activated, cis - recombination occurs in the correct forward direction in the EEVs, and they are expressed in a dose - dependent manner. The Cre recombinase is under the control of the TRE promoter (see below). Dox binds to the reverse tetracycline - controlled trans - activator (rtTA) to form the Dox - rtTA complex. The Dox - rtTA complex binds to the tetracycline - responsive element (TRE) and activates the downstream target gene (e.g., EEV) that becomes correctly oriented only during Cre recombination events. The inhibitor, reverse tetracycline - controlled trans - silencer (rtTS), serves two functions: (1) minimizing the leaky expression of the TRE promoter and (2) enhancing rtTA activity in the presence of doxycycline, which functions as a co - activator. Other abbreviations: Cbh, CMV, and chicken beta - actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 polyA tail.

[0144] Example 4. Generation of a lentiviral packaging cell line (fHEK - LV) A vector containing the Gag, Pol, Tat, and Rev (GPTR) gene cassettes along with the Bsd selection marker (see Figure 4A) is transfected into fusion HEK 293F (fHEK) cells by electroporation using a Bio - Rad Gene Pulser. Vector DNA and the Piggy Bac transposase vector are added to the cell suspension, and electroporation is performed using a single - pulse exponential decay. The cells are grown in a medium containing the selection concentration of blasticidin, and the surviving cells (designated fHEK - GPTR) are grown and prepared for transfection of the VSV - G - inducible gene cassette.

[0145] Figure 4A: Gag, Pol, Tat, and Rev (GPTR) integration gene cassette. The GPTR gene cassette is adjacent to the left and right Piggy Bac transposon inverted terminal repeats (LTR and RTR, respectively). CAG is a hybrid promoter composed of the CMV early enhancer element, the chicken beta-actin promoter, and the rabbit beta-globin gene splice acceptor. pA, SV40 polyA tail; Bsd, blasticidin resistance gene.

[0146] A vector containing the VSV-G-inducible gene cassette (Figure 4B) is transfected into fHEK-GPTR by electroporation using a Bio-Rad Gene Pulser with a single-pulse exponential decay. The transfected cells are cultured in selective medium containing blasticidin and hygromycin.

[0147] Figure 4B: Inducible cassette for the activation of the envelope VSV-G gene and the gene encoding Cre recombinase. The inducible cassette is flanked by Sleeping Beauty transposon inverted terminal repeats (left and right, abbreviated as LTR and RTR, respectively). VSV-G is in the reverse orientation and flanked by heterologous Lox (LoxP and Lox511) sites. Cre recombinase is under the control of the TRE promoter (see below). When doxycycline is activated, Cre is expressed, cis-recombination occurs in the correct forward orientation in VSV-G, and VSV-G is activated in a dose-dependent manner. Dox binds to the reverse tetracycline-controlled transactivator (rtTA) to form the Dox-rtTA complex. The Dox-rtTA complex binds to the tetracycline response element (TRE) and activates the downstream target gene. The inhibitor, reverse tetracycline-controlled transsilencer (rtTS), serves two functions: (1) minimizing the leaky expression of the TRE promoter and (2) enhancing rtTA activity in the presence of doxycycline, which functions as a co-activator. Other abbreviations: Cbh, CMV, and chicken beta-actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 polyA tail.

[0148] Example 5. Generation of a lentiviral packaging cell line (fHEK-SLV) containing the GPTR, inducible envelope, and STAC gene cassette The vector containing the VSV-G and STAC cassettes (Figure 5) is transfected into fHEK-GPTR by electroporation using a Bio-Rad Gene Pulser with a single-pulse exponential decay. The transfected cells are cultured in selective medium containing blasticidin and hygromycin.

[0149] Figure 5: Inducible STAC cassette for the amplified synergistic activation of the VSV-G gene and the packaging genes Gag, Pol, Tat, and Rev (GPTR, see Figure 4). The inducible cassette is flanked by Piggy Bac transposon inverted terminal repeats (abbreviated as left and right LTR and RTR, respectively). The transcriptional co-activator and histone acetyltransferase (HAT), p300, and the HIV-1 Tat transcriptional activator both bind to multiple GCN4 peptides (shown as black squares). Both fusion proteins are expressed under the control of the CMV promoter. The hybrid scaffold SSPVP consists of an scFv fragment that binds to the GCN4 peptide, and the transactivation domains of p53, VP64, and p65 to recruit additional transcription factors, HAT, and co-activator to the basal transcriptional apparatus at the promoter.

[0150] Inducible cassette for the activation of the envelope VSV-G gene and the gene encoding Cre recombinase. VSV-G is in the reverse orientation and flanked by heterologous Lox (LoxP and Lox511) sites. When doxycycline is activated, Cre is expressed and cis-recombination occurs in the correct forward orientation in VSV-G, activating VSV-G gene expression in a dose-dependent manner. When doxycycline is induced, Dox binds to the reverse tetracycline-controlled transactivator (rtTA). The Dox-rtTA complex binds to the tetracycline response element (TRE) and activates the downstream target gene. The inhibitor, reverse tetracycline-controlled transsilencer (rtTS), serves two functions: (1) minimizing leaky expression of the TRE promoter and (2) enhancing rtTA activity in the presence of doxycycline, which functions as a co-activator. Other abbreviations: Cbh, chicken beta-actin and CMV hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 polyA tail.

[0151] 5.1 Other possible gene modifications and potential benefits Chromatin modifications can contribute both positively and negatively to gene transcription. In actively transcribed genes, histone acetylation is abundant both at the promoter and in the 5' region of the gene's coding region. Among the currently known enzyme histone acetyltransferases (HATs), CBP / p300 globally acetylates thousands of sites, many of which are signature histone sites in active gene transcription regions (Weinert et al., 2018). p300 / CBP is often referred to as a single entity due to its extensive homology and functional similarity (reviewed in Kouzarides 2007). p300 / CBP also functions as a transcriptional co-activator, a protein that bridges (bridges) transcriptional activators and components of the basal transcription apparatus (reviewed in Janknecht and Hunter, 1996). P300 functions as a transcriptional co-activator for a broad repertoire of signaling pathways, including the HIV-1 viral machinery and adenovirus E1A recruitment.

[0152] The benefits of enhanced virus production can be obtained by increasing the exogenous expression of global HATs such as p300 / CBP, or by attenuating HDAC, an active competitor of p300. HDAC deacetylates the same sites as p300 / CBP (Li et al., 2014).

[0153] Example 6. Generation of a packaging cell line with exogenous expression of p300 A vector containing the coding region of p300 expressed under the control of the CMV promoter and the blasticidin resistance gene (Figure 6A) is transfected into fused HEK 293F (fHEK) cells by electroporation using a Bio-Rad Gene Pulser using a single-pulse exponential decay. Selection medium containing blasticidin is added to the medium, and viable cells containing stable integration of p300 are grown.

[0154] Figure 6A is a diagram of the p300 integration gene cassette. The p300 gene is adjacent to the CMV promoter and the SV40 polyA tail. The blasticidin (Bsd) resistance gene cassette is located downstream of the p300 gene cassette containing the CMV promoter, the gene encoding Bsd, and the SV40 polyA tail. Figure 6B shows the HDAC shRNA gene cassette. The HDAC shRNA is adjacent to the U6 promoter for shRNA expression. The blasticidin (Bsd) resistance gene cassette is located downstream of the shRNA expression cassette containing the CMV promoter, the gene encoding Bsd, and the SV40 polyA tail. Figure 6C shows the spliced Xbp1 (Xbp1s) gene cassette. The Xbp1s gene is adjacent to the CMV promoter and the SV40 polyA tail. The blasticidin (Bsd) resistance gene cassette is located downstream of the Xbp1s gene cassette containing the CMV promoter, the gene encoding Bsd, and the SV40 polyA tail.

[0155] Example 7. Generation of a packaging cell line with knockdown of HDAC Lentivirus packaging is performed by complexing the U6-HDAC shRNA transfer vector (Figure 6B) with the helper vector, packaging vector, and envelope vector using polyethyleneimine. The complex is added to HEK293 cells. The supernatant is collected after transfection, and the lentivirus titer is measured by sandwich ELISA. fHEK is infected with lentivirus particles containing U6-HDAC shRNA. The transduced cells are grown in selective medium containing blasticidin, and the surviving cells containing stable integration of U6-HDAC shRNA are grown.

[0156] Further enhancement of the endoplasmic reticulum (ER) can be achieved by activation of the unfolded protein response (UPR) pathway. Under ER stress, unspliced Xbp1 mRNA is cleaved by the activated stress sensor Ire1α, generating spliced Xbp1 (Xbp1s). The translated Xbp1s translocates to the nucleus of hundreds of target genes encoding ER molecular chaperones, folding enzymes, and ER-associated protein degradation (ERAD). Enhanced Xbp1 can increase ER biosynthesis in mammary epithelial cells (Sharmin et al., 2021). The benefits of enhanced ER function by increasing the expression of spliced Xbp1 can be exploited for increased virus production.

[0157] Example 8. Generation of a packaging cell line with exogenous expression of spliced Xbp1 A vector containing the coding region of spliced Xbp1 (Xbp1s) expressed under the control of the CMV promoter and the blasticidin resistance gene (see Figure 6C) is transfected into fusion HEK 293F (fHEK) cells by electroporation using a Bio-Rad Gene Pulser with a single pulse exponential decay. Selection medium containing blasticidin is added to the medium, and the surviving cells contain stable integration of Xbp1.

[0158] Example 9: Selection protocol for fused cells with high mitochondria and high ROS To screen for different phenotypes and reactive oxygen species (ROS) of mitochondria, the cells were stained with CellROX® Deep Reagent, a fluorescent probe for measuring cellular oxidative stress in cells, TMRM (tetramethylrhodamine methyl ester) for measuring the mitochondrial membrane potential in live cells, and Biotracker405 Blue Mitochondria for staining the mitochondrial membrane. In this experiment, LIVE / DEAD Fixable NIR was used to stain live cells.

[0159] Figure 8 shows the workflow used. Fusion cells were cultured in a complete, animal - origin - free (AOF), chemically defined cell culture medium: CDM4 PerMAb + 6 mM L - glutamine and detached using StemPro™ Accutase™ cell dissociation reagent. The cell sample was combined with a calculated amount of each staining dye to a final concentration of approximately 200 mL of cell suspension containing 1×10 8 cells in a 500 mL shaking flask. The samples were incubated overnight at 120 rpm, 37 °C in a shaker with 8% CO2.

[0160] For cell sorting, 200 mL of the cell sample was centrifuged at 300×g for 5 minutes. The cell pellet was resuspended in Accutase cell dissociation reagent, diluted, and strained into a sterile 50 mL centrifuge tube. Cells were sorted using a Sony SH800S cell sorter with the following gates: Gate 1 - cell ID gate; Gate 2 - singlet gate; Gate 3 - live cell gate; Gate 4 - Biotracker405Blue mitochondria (select top 10%); Gate 5 - TMRE×CellROX Deep Red (select top 10% quadrant).

[0161] A population of 500,000 sorted cells was grown for 4 - 5 days and used for single - cell cloning in a 96 - well plate containing 150 μl of medium per well. When individual wells became 80% confluent, they were grown step - by - step and transferred into a 125 mL shaking flask and used to create a cell bank.

[0162] Example 10. Protocol for Transient Transfection of HEK291 Cells to Express AAV Capsids Containing Reporter Genes Useful sources of reagents are the AAV - MAX system available from ThermoFisher, the VirusGEN® AAV transfection kit, and the AAVpro helper - free system for various serotypes of AAV from Takara.

[0163] The day before transfection, the host cells were placed in 25 mL of a complete, animal - origin - free (AOF), chemically defined cell culture medium (CDM4 PerMAb) containing 6 mM L - glutamine, at a density of 3×10 6 viable cells / mL in a 125 mL cell culture flask and grown to a density of 3.0×10 6 viable cells / mL (the viability was over 90%). The cells were centrifuged at 300×g in a conical tube and resuspended in 25 mL of virus production medium. After transferring to a new shaking flask, 250 μL of AAV - MAX Enhancer was added. The cells were cultured on an orbital shaker in a 37°C incubator until the formation of the DNA / transfection complex was complete.

[0164] Figure 8 maps the plasmids used to generate an AAV vector containing a highly sensitive green fluorescent protein (EGFP) reporter gene for the purpose of screening cell hybrids for virus production. A mixture of these plasmids was prepared as shown in Table 4. [Table 4]

[0165] 75 μL of Trans - ITVirusGEN® was added to the plasmid in the AAV CFS&E solution and incubated at room temperature for 15 minutes to form the transfection complex. 2.7 of this mixture was added to each 125 mL flask and placed in the incubator again for 12 - 18 hours. 0.3 mL of 0.5 M sodium butyrate was added, and the cells were harvested 72 hours after transfection.

[0166] Incorporation by reference For all purposes in the United States of America, each publication and patent document referred to in this disclosure is incorporated by reference in its entirety for all purposes to the same extent as if each publication or document were specifically and individually indicated to be incorporated by reference herein.

[0167] The implementation of the invention described in the claims The technology and its use provided in this disclosure are described within the scope of a hypothetical understanding of the general principles of virus and pharmaceutical manufacturing. These descriptions are provided for the enlightenment and interest of the reader and are not intended to limit the implementation of the invention described in the claims. All products and methods described in the claims of this application may be used for any suitable purpose without limitation, unless otherwise specified or required.

[0168] Although this disclosure is described with reference to specific embodiments, modifications can be made to adapt this disclosure to specific situations or intended uses as routine experimental matters, and equivalents can be substituted, so as to achieve the benefits of this disclosure without departing from the scope of the claims.

Claims

**Claim 1** A method for producing a viral vector or particle, comprising: providing a starter population of cultured cells; forming cell hybrids, each containing two or more cells, from the starter population; genetically modifying the cell hybrids to express a viral element and a payload; culturing the genetically modified cells to produce the viral vector or particle containing the element and encapsulating the payload; A method comprising the above steps. **Claim 2** A process for establishing a production cell line for the high-efficiency production of a viral vector or particle, comprising: (a) providing a starter population of cultured cells; (b) forming cell hybrids, each containing two or more cells, from the starter population; (c) dividing the cell hybrids into a plurality of aliquots; (d) collecting cell samples from each aliquot; (e) genetically modifying the cells in each sample to express a viral element and a reporter gene; (f) measuring the production of a viral vector or particle containing the product of the reporter gene by each of the samples, thereby identifying which aliquot contains cell hybrids that produce a viral vector or particle with a higher functional titer than the cell hybrids in other aliquots; (g) growing cells from one or more of the aliquots identified in step (f) to establish the production cell line. A process comprising the above steps. **Claim 3** The process according to claim 2, wherein the cell hybrids formed in step (b) are cloned in step (c), or the cells measured in step (f) are cloned during or after step (g). **Claim 4** The process according to claim 2 or 3, wherein the reporter gene encodes a protein that emits a detectable signal, such as a highly sensitive green fluorescent protein (EGFP). **Claim 5** The process according to any one of claims 2 to 4, wherein step (c) comprises sorting or separating cell hybrids according to cell phenotype. **Claim 6** The process according to claim 5, wherein the cellular phenotype comprises one or more intracellular organelles, such as mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, or nucleosomes, and / or one or more cytoplasmic or intracellular characteristics, such as high or low intracellular content of reactive oxygen species (ROS), cellular redox, or pH.

7. The process according to claim 5, wherein the cellular phenotype comprises a combination of high levels of mitochondria and ROS per cell.

8. The process according to any one of claims 2 to 7, wherein step (f) comprises identifying which aliquot contains cell hybrids that produce more virus capsids per cell.

9. The process according to any one of claims 2 to 8, wherein step (f) comprises identifying which aliquot produces a higher proportion of capsids filled with the reporter gene.

10. The method or process according to any one of the preceding claims, further comprising selecting cell hybrids or cell aliquots having a higher growth rate than other cell hybrids.

11. The method or process according to the preceding claims, wherein the starter population is a single cell line.

12. The starter population is a cell line selected from CHO cells, mouse myeloma NSO cells, mouse myeloma SP2 / 0 cells, human embryonic kidney 293 (HEK293) cells, baby hamster kidney 21 (BHK-21) cells, VERO cells, PER.C6 cells, and HeLa cells, their autotypic hybrids, and hybrids of such cells with cells or primary cells derived from another cell line, The method or process according to any one of the preceding claims.

13. A process for establishing a production cell line for high-efficiency production of virus vectors or particles, comprising: (a) providing a starter population of cultured cells; (b) forming cell hybrids from the starter population, each cell hybrid containing two or more cells; (c) dividing the cell hybrids into a plurality of aliquots; (d) testing the aliquots or subpopulations thereof to identify which aliquot has a greater amount of mitochondria and / or reactive oxygen species (ROS) per cell; (e) growing cells derived from one or more of the aliquots identified in step (d) to establish the production cell line; A process comprising the same. **Claim 14** The process according to any one of claims 2-9 or claim 13, further comprising genetically modifying cells derived from the production cell line to express a transgene encoding one or more viral elements, whereby the cells are adapted to produce a viral vector or particle containing the element. **Claim 15** The process according to claim 14, further comprising genetically modifying the cells to express a transgene encoding a payload, whereby the cells are adapted to produce a viral vector or particle encapsulating the payload. **Claim 16** A production cell line adapted for high-efficiency production of viral vectors or particles, established according to the process of any one of claims 2-9 and 13-15. **Claim 17** The production cell line according to claim 16, which is genetically modified to express a transgene encoding one or more viral elements, whereby the cells are adapted to produce a viral vector or particle containing the element. **Claim 18** A production cell line, wherein each cell is a hybrid of two or more parental cells derived from a starter cell population, and the production cell contains an expressible transgene encoding one or more viral elements, whereby the cell is configured to produce a viral vector or particle containing the element. **Claim 19** The production cell line according to claim 17 or 18, wherein the cell further contains a transgene that constitutes or encodes a payload, whereby the cell is adapted to produce a viral vector or particle encapsulating the payload. **Claim 20** The production cell line according to claim 19, wherein the payload comprises a nucleic acid configured to be expressed in vivo in the subject when administered to a human subject. **Claim 21** The production cell line according to claim 19, wherein the payload comprises a protein configured to be delivered to the cells of the subject when the particle is administered to the subject. **Claim 22** The production cell line according to claim 19, wherein the payload comprises a nucleic acid or a protein configured for ex vivo transfection of cells.

23. The production cell line according to any one of claims 18 to 22, wherein at least 500 or 5,000 virus vectors or particles are produced per cell by culturing the production cells.

24. The production cell line according to any one of claims 18 to 23, which produces more virus vectors or particles, produces virus vectors or particles having a higher specific activity, or produces virus vectors or particles having a higher proportion of the payload, as compared with cells derived from the starter cell population genetically modified with the same transgene.

25. A production method comprising culturing cells derived from the production cell line according to any one of claims 19 to 24 to produce virus vectors or particles encapsulating the payload.

26. The method according to claim 1 or 25, further comprising purifying the vector or particle from the cell culture and formulating the vector or particle to produce the pharmaceutical product in a manner suitable for administration to humans.

27. A pharmaceutical product for administration to a human subject, obtained according to the method of claim 25 or 26.

28. A therapeutic product configured for ex vivo treatment of a human subject in need thereof and obtained according to the method of claim 25.

29. A treatment method comprising administering the pharmaceutical product according to claim 27 to a subject in need thereof.

30. An ex vivo treatment method comprising contacting a plurality of cells that are autologous or allogeneic to the subject with the therapeutic product according to claim 28, and then administering the cells to the subject.

31. The method, process, or product according to any one of the preceding claims, wherein the virus vector and / or virus particle is an adeno-associated virus (AAV) vector or particle.

32. Use of the method, process, or product according to any one of claims 1 to 26 in the production of virus vectors or particles for the treatment of human diseases or conditions.

33. The method, process, or product according to any one of claims 1, 15, 19 to 22, and 24 to 25, wherein the payload is a polynucleotide encoding a gene product for gene therapy of a human subject in need of gene therapy.

34. The method, process, or product according to any one of claims 1, 15, 19 to 22, and 24 to 25, wherein the payload is a target antigen of a pathogenic microorganism or a nucleic acid encoding the target antigen for inducing an immune response against the pathogenic microorganism in a subject in need of an immune response.

35. The method, process, or product according to claim 34, wherein the microorganism is SARS-CoV-2, a virus that causes COVID-19.

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