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

Abstract

The present disclosure provides a technique for adapting host cells to increase the yield and improve the quality of viral vectors and particles. The cell hybrids are formed from a parent cell line and are divided or cloned into a plurality of aliquot samples for testing. An aliquot sample having high throughput and phenotypic characteristics for viral production, such as an optimal level of intracellular organelles, is selected and used to establish a production cell line. The producer cell may be genetically engineered to express a transgene encoding a viral element to produce a viral vector or particle having a therapeutic payload. Each of the hybridized producing cells produces more viral vectors or particles and has a higher functional titer, thereby reducing the production costs of agents for gene therapy and immunization.

Description

Related Applications

[0001] This patent disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 350,863, filed on June 9, 2022. The contents of the priority application are hereby incorporated by reference in their entirety for all purposes. Technical Field

[0002] The present disclosure generally relates to the production of pharmaceutical compounds containing viral components. It also relates to the modification, selection and genetic engineering of host cells to produce high-level pharmaceutical products with improved biological and pharmacological properties. Background Art

[0003] Over the past decade, viral vector-based therapies have become a real option in clinical medicine. The FDA has approved more than a dozen therapies using viral vectors, covering three different types of viral vectors: adeno-associated virus (AAV), lentivirus, and herpes simplex virus. Adenoviral vectors have been approved as immunogenic compositions for the treatment of infectious diseases such as COVID19. There are currently about 25 viral vector therapies in late-stage development and another 120 in Phase II trials, and the number of viral vectors approved for commercial production will increase rapidly (E. Capra et al., McKinsey and Company, 2022).

[0004] The first gene therapy vectors are typically developed to treat rare diseases. There is growing interest in treating more common diseases, which requires higher production volumes and lower cost of goods. Over the past few years, large contract development and manufacturing organizations (CDMOs) have invested billions of dollars in viral vector production facilities. This burgeoning interest is promising, but the rapid influx of funding and the development of new technologies have not solved the bottlenecks and challenges of viral vector manufacturing.

[0005] Currently, lack of standardization and low yields are part of the challenge. Physical properties and functional requirements vary widely between different carriers. A high degree of process optimization is still required for each product. Low recoveries in the chromatography steps mean that yields are often less than 50% (M. May, Biotech. Eng. News, August 2, 2021). In contrast, and The manufacturing of therapeutic antibodies and biosimilars is performed using standardized platforms and typically achieves yields above 90%.

[0006] The owner of the technology described in this disclosure previously developed a system for increasing the yield of monoclonal antibodies in production cell lines by more than four times. U.S. Patent No. 10,329,594. Cultured cells (e.g., CHO cells) are fused together and hybrids with high levels of endoplasmic reticulum or Golgi are selected. Unfortunately, the intracellular machinery required to make viral vectors is completely different. Most viruses replicate in the cytoplasm, not in the ER, and must be coordinated within the cell to achieve correct vector assembly. Summary of the invention

[0007] The present disclosure provides a technology for adapting host cells to maximize production and improve the quality of viral vectors and particles. Cell hybrids are formed by parental cell lines and are divided or cloned into multiple aliquots for testing. Aliquots with high production capacity and phenotypic characteristics for viral production (e.g., optimal levels of intracellular organelles) are selected and used to establish production cell lines. Production cells can be genetically modified to express transgenics encoding viral elements to produce viral vectors or particles with therapeutic payloads. Each cell of the hybrid production cell produces more viral vectors or particles, with higher functional titers, thereby reducing the production cost of medicaments for gene therapy and immunity.

[0008] In general, the techniques proposed in this disclosure can be used to produce viral vectors or particles. The system is implemented by providing a starting population of cultured cells; forming cell hybrids from the starting population, each cell hybrid comprising two or more cells, and genetically modifying the cell hybrids to express elements of the virus and drug payload. The cells are then cultured to produce viral vectors or particles containing the elements and encapsulating the drug payload.

[0009] Throughout this disclosure, the terms "fused cells," "cell hybrids," and "engineered cells" refer interchangeably to cells made by combining two or more parental cells to produce a single cell with organelles and chromosomes from all parents within the combined plasma membrane. A "payload" is a polypeptide, polynucleotide, or any other compound or composition that is encapsulated in a viral capsid or other macromolecular package. Exemplary payloads are reporter genes for cell screening or therapeutic payloads for medical treatment. A "viral vector" is a capsid that is configured to cause expression of the nucleotides it contains when administered in vivo. A "viral particle" is a capsid that is configured to deliver a protein or other payload to a cell when administered in vivo. A "genetically altered" cell contains an expressible transgene, either within the genome of the cell or as a plasmid that is expressed elsewhere in the cell.

[0010] One way of implementing the technology disclosed herein is to create a production cell line library, which can be used to manufacture capsids containing different useful loads. By providing a starting population of cultured cells, a cell hybrid is formed from the starting population, and each hybrid comprises the contents of two or more parental cells to manufacture a production cell line for efficient production of viral vectors or particles. The hybrid is distributed in a plurality of aliquots, which are then sampled for testing. The sample is genetically modified to express one or more transgenics encoding viral system elements, and a reporter gene (reporter) for determining the efficiency of viral components or capsids produced by genetically modified cells in each aliquot. The reporter gene can be a viral component itself or a drug useful load. In order to screen, it is convenient to use a reporter gene encoding a gene product that is easy to express, such as a protein that produces a light signal, such as green fluorescent protein or fluorescein.

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

[0012] After transfection, the user characterizes each sample by measuring the yield and / or quality of the viral vector or particles containing the reporter gene product for each sample. Aliquots containing cells with the desired properties are expanded in culture to establish production cell lines. Optionally, the user can perform one or more additional aliquoting, characterization, and amplification cycles to further enrich for cells with the desired phenotype and / or viral production capacity. If desired, cells can be cloned at any time in the process: either as a means of aliquoting cells at the beginning or to stabilize cell properties in subsequent steps.

[0013] Alternatively or in addition, aliquots of cells can be separated or selected based on empirically determined cellular properties associated with favorable virus production. For example, this can be done as part of an aliquoting process where cells are sorted or separated based on any combination of high or low intracellular content of one or more organelles (e.g., mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, or nucleolus) and / or one or more cytoplasmic or transcellular features (e.g., reactive oxygen species (ROS), cellular redox, or pH). Phenotypic separation or selection can be performed sequentially or simultaneously with the evaluation of virus production, so the final cell selection may be a combination of phenotype and production capacity.

[0014] Identification and selection of aliquots or clones to be amplified can be based on which aliquots contain cell hybrids that produce more viral capsids per cell, or which aliquots produce capsids with a greater proportion of reporter genes filled, or a combination of both. Depending on the user's purpose, the technology of the present disclosure can be implemented by using a single cell line as a starting cell population, 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; using their autotypic hybrids; or using a combination or hybrid of these cells with cells from another cell line or primary cells.

[0015] In order to produce viral vectors and particles for treatment or other purposes on an industrial scale, cells from established cell banks are genetically modified to express transgenes encoding one or more elements of the virus, as well as transgenes constituting or encoding the desired payload. Transfection can be transient or stable (permanent). Transgenes for viruses and payloads can be inserted into the genome of production cells transiently or stably in various combinations. For example, production cell lines can be stably transfected with the components required for virus assembly to establish production cell lines dedicated to the selected virus type. Dedicated production lines can then be transiently transfected to contain different payloads depending on clinical goals.

[0016] The present disclosure includes production cell lines suitable for efficient or high-quality production of viral vectors or particles established according to any of the above methods and procedures. Optionally, the production cell line can be genetically modified with a plasmid encoding one or more elements of the virus (e.g., Rep and Cap genes of AAV), and optionally with a helper plasmid (e.g., encoding adenoviral genes that promote AAV packaging), so that the cell is adapted to produce a viral vector or particle containing the element. Some production cells of this technology can be characterized as a hybrid of two or more parental cells from a starting cell population, wherein the production cell contains an expressible transgene encoding one or more elements of the virus, so that the cell is configured to produce a viral vector or particle containing the element.

[0017] Such production cells may also contain a transgene that constitutes or encodes a payload, thereby adapting the cell to produce a viral vector or particle that encapsulates the payload. Embodiments of such payloads include any one or more of the following: (1) a nucleic acid that is configured to be expressed in a human subject after administration to the subject; (2) a protein that is configured to be delivered to a subject's cells after administration of the particle to the subject; (3) a nucleic acid or protein that is configured to be transfected into a cell ex vivo; and / or (4) a reporter gene as described above.

[0018] Any production cell disclosed herein can produce a specified number of viral vectors or particles per cell, as described below. Alternatively or additionally, the production cells disclosed herein (compared to cells from a starting cell population that has been genetically modified with the same transgene) can be characterized as producing more viral vectors or particles; producing viral vectors or particles with higher functional titers; and / or producing viral vectors or particles with a higher proportion of a specific payload filled, as described below.

[0019] The present disclosure provides methods for manufacturing viral vectors or particles that encapsulate payloads, such as by culturing cells from established production cell lines of the present disclosure. The viral vectors or particles thus produced can be formulated as pharmaceuticals under GMP conditions to produce a drug product suitable for human administration. The viral vectors or particles thus produced can also be configured for ex vivo treatment of human subjects in need.

[0020] The present disclosure provides a method of treatment by administering the above-mentioned viral vectors or particles to a subject. Alternatively, the vectors and particles can be used for ex vivo treatment by contacting them with a plurality of cells (usually autologous or allogeneic to the subject) and then administering the cells to the subject.

[0021] An exemplary viral system that can be used in any aspect of the present disclosure is an adeno-associated virus (AAV) vector or particle and selected serotypes thereof. In principle, the technology of the present disclosure can be used to establish a production cell line for any viral system, such as adenovirus, alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.

[0022] The viral vectors manufactured according to the present disclosure can be used to treat a variety of human diseases or conditions. The payload can be a polynucleotide encoding a gene product for gene therapy of a human subject in need. The payload can be a target antigen of a pathogenic microorganism (e.g., SARSCoV2), or a nucleic acid encoding the target antigen, for eliciting an immune response against the microorganism in a subject in need.

[0023] Various aspects, embodiments, features and characteristics of the present invention are described in the following section, drawings and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a workflow for obtaining hybrid cell lines with high virus production capacity. Cells are fused together to produce an engineered cell population. The cell population is then cloned or divided into separate aliquots, optionally taking into account the desired cell phenotype (e.g., intracellular organelle content). Individual clones or aliquots are transiently transfected to express viral elements and reporter proteins, and high-yield cells are identified. Selected cells are amplified to establish a production cell line bank. Production cells can be transfected to express viral vectors or particles that encapsulate therapeutic payloads for gene therapy or immunity.

[0025] Figure 2 It was shown that the protein production of the CHO cell populations fused and selected for high endoplasmic reticulum content was increased 4.5-fold compared to the parental CHO cells. The cell populations were all transfected to express secreted alkaline phosphatase (SEAP).

[0026] Figure 3A is a genetic map of the Rep-Cap inducible integration gene cassette for activating E2A, E4 and VA RNA (abbreviated as EEV and Cre recombinase). Figure 3B depicts the inducible cassette for activating E2A, E4 and VA RNA (abbreviated as EEV and Cre recombinase). Figure 3C The inducible STAC cassette used to amplify the synergistically activating E2A, E4 and VA RNA (EEV) was mapped.

[0027] Figure 4A is a genetic map of the Gag, Pol, Tat and Rev (GPTR) integration gene cassettes. Figure 4B depicts the induction cassettes used to activate the envelope VSV-G gene and the gene encoding the Cre recombinase.

[0028] Figure 5 It is a genetic map for amplifying the inducible STAC cassette that co-activates the VSV-G gene and the packaging gene.

[0029] Figure 6A is a schematic diagram of the p300 integrated gene cassette. The p300 gene is flanked by a CMV promoter and a SV40 poly A tail. Figure 6B shows the HDAC shRNA gene cassette. The HDAC shRNA is flanked by a U6 promoter for shRNA expression. Figure 6C shows the spliced ​​Xbp1 (Xbp1s) gene cassette.

[0030] Figure 7 Shown is the workflow for screening confluent HEK 293 cells based on their intracellular mitochondrial and reactive oxygen species (ROS) content.

[0031] Figure 8 It is a set of gene maps for the preparation of plasmids containing the enhanced green fluorescent protein (EGFP) reporter gene of AAV vectors, which are used to screen cell hybrids for virus production.

[0032] Fig. 9A The workflow for producing AAV vectors using suspension HEK 293 cells as hosts is shown. Left: Cell seeding, transfection, and harvesting of AAV particles from lysed cells. Right: Product quality assessment: capsid quantification, viral titer (or capsid concentration), percentage of full capsids, and transduction function titer. Fig. 9B Shown are the numbers of AAV2 viral particles that can be produced from confluent HEK 293 cells.

[0033] Fig. 10A , 10B Figures 10 and 10C show higher capsid productivity from several serotypes of AAV vectors in confluent HEK 293 cells. The engineered clones (17-2) showed a 3-fold, 9-fold, and 2.5-fold increase in AAV vector production compared to the parental host, respectively.

[0034] Fig.11A and 11B Higher functional titers of several serotypes and other features of AAV vectors from confluent HEK 293 cells were shown.

[0035] Fig.12The results show an increase in the proportion of capsids containing nucleic acid payloads achieved by screening the cellular phenotype of fused cells. An engineered HEK 293T pool (4C1) clone (#40) selected for high mitochondrial (top 10%) and high reactive oxygen species (top 10%) showed a two-fold increase in the full-to-empty ratio for the AAV2 serotype. DETAILED DESCRIPTION

[0036] The present disclosure provides an improved cell line for making a medicament containing viral elements, greatly reducing the cost of commercial production. Cell lines are obtained by selecting cells from a mixed population to obtain one or more characteristics of supporting virus or non-specific virus production, such as the level of mitochondria, endoplasmic reticulum, Golgi apparatus and / or other desired phenotypic characteristics, compared to other cells in the starting mixture. Cells prepared for functional selection by preparing cell hybrids can obtain particularly effective production cell lines. Expressible genes containing expected viral vectors or particle elements can be transfected into cells before or after one or more fusion and selection cycles. 1. Overview

[0037] Figure 1 is a general scheme outlining a suitable workflow in which the disclosed technology may be implemented.

[0038] Cells from selected sources (e.g., established cell lines) are fused together in multiple cycles to produce heterogeneous hybrid populations in the ability to synthesize viral vectors or particles. In order to obtain high-yield cells from a population, the population is divided into multiple separate aliquots or clones. As part of the classification (partitioning), cells can be screened or separated based on specific phenotypic features that are known or suspected to be conducive to high-level viral capsid production or filling. Samples from each separate aliquot or clone are tested individually to determine their ability to produce a large amount or high titer vectors or particles. Aliquots that have now been shown to be high-yield cells are amplified and used to establish one or more production cell lines.

[0039] Then, the stored (banked) production cells can be obtained for industrial-scale production of one or more therapeutic viral vectors or particles, particularly using viral components of the same species and serotype used for screening. The cells selected for industrial production can be transfected in the same manner as screening, except that the reporter gene is replaced with a therapeutic payload. The final transfection can be transient, or the viral elements can be integrated into the genome of the production cell with an inducible promoter, and then different payloads can be packaged into the same viral system by transient transfection.

[0040] Other workflows may be effective, depending on the choice of viral system and the user's goals. Various aspects of the disclosed technology can be practiced in any combination and in any order to effectively generate the viral vector or particle desired by the user. 2. Advantages of this technology

[0041] Depending on the practice and mode of application, the aspects of the present disclosure described in this disclosure can be used to select cell hybrids that produce viral vectors and particles at higher functional titers per volume of culture fluid. This in turn has the following advantages. Reduce the production costs of viral vectors and particles for clinical use, thereby improving access to such therapeutics; Reduce the need to expand or build new GMP manufacturing facilities as market size increases; Provide GMP production of kilogram quantities of finished protein using relatively small or fewer bioreactors, ·Create established production cell lines suitable for high-level expression of different vector and vaccine families as needed; Reducing the number of cloning or selection steps required after integration of the gene to be expressed; Improve product quality; and Provide high-quality, low-volume research materials, thereby reducing the time required to initiate and complete clinical trials.

[0042] The technology disclosed herein can also be used to increase the load of the viral capsids produced, thereby increasing the effective titer of the preparation. Prior to this discovery, most of the AAV capsids produced by host cells were empty (10-30% full capsids are the industry average), which adversely affects the efficacy and safety of AAV drug products and increases the risk of immunogenicity. The following provides a technique for selecting cells with higher mitochondrial and reactive oxygen content, which results in a two-fold increase in the proportion of capsids carrying the payload. 3. Principle

[0043] The disclosed techniques for packaging vectors such as lentivirus, adenovirus and adeno-associated virus (AAV) exploit the cellular compartment dependence of key proteins involved in viral packaging and replication.

[0044] Physical associations between the endoplasmic reticulum (ER), mitochondria, peroxisomes, and nucleoli have been implicated in the production of viral proteins. MAMs, sites of transient contact between the ER and mitochondria, provide calcium microdomains for cellular signaling, such as activation of Ca++-dependent metabolic enzymes. Evidence for ER-to-mitochondrial trafficking has been associated with the human cytomegalovirus UL37 protein, where highly conserved sequences drive translocation of these proteins to the ER.

[0045] Viral mitochondrial localized inhibitor of apoptosis (vMIA) works in both the ER and mitochondria. In addition to viral proteins, viral RNA can also target mitochondria (Reeves et al., 2007). Other key viruses that are essential for ER and / or mitochondrial transport include hepatitis C virus (HCV) and related viruses of Flaviviridae, polyomavirus, rotavirus, coronavirus, poliovirus, enterovirus, hepatitis E virus, HIV-1, adeno-associated virus (AAV; Williamson et al., 2012; Ravindran et al., 2016). Adenovirus E1B19K packaging protein is required for AAV viral packaging and is located in mitochondria.

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

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

[0048] The technology disclosed herein further enhances the manufacture of viruses by utilizing subcellular organelles (e.g., endoplasmic reticulum, mitochondria, peroxisomes, and nucleoli) to create super manufacturing cell lines. These benefits are achieved by engineering cell lines to select high ER (including enhanced unfolded protein response), mitochondrial content, peroxisomes, or properties of single or multiple nucleoli, as discussed in the present disclosure. Virus yield is increased by selecting cells with higher levels of subcellular machinery or biochemistry from a mixed cell population compared to other hybrids or parental cells in the starting mixture, thereby supporting increased virus yield.

[0049] At least one of the selected phenotypic characteristics is not necessarily specific to the production of a particular virus. The characteristic is not simply the expression level of the protein or surrogate of interest. Rather, it is a characteristic that supports the production of a variety of different viruses. These characteristics include the relative density of subcellular organelles, particularly those involved in viral packaging, and the relative levels or concentrations of enzymes that contribute to the packaging of a variety of different proteins, such as HIV, adenovirus, and adeno-associated virus (AAV).

[0050] Viral yields can be further enhanced by stable gene copy amplification of viral genes critical for packaging and replication, histone modifiers, protein biosynthetic pathway enhancers, and transcriptional amplifiers, thereby creating unique manufacturing cell lines that may be able to amplify viral yields tens to hundreds of times compared to traditional host cell lines used for viral production in the industry, as described in the present disclosure. 4. Technical methods

[0051] Repeated homotypic fusion of viral production cells (e.g., HEK 293 cells) is thought to result in genome shuffling and amplification of entire chromosomes within a single cell. Selection of cells with the desired phenotype can improve manufacturing capacity. Fusion cell hybrids are more suitable for the production of viral vectors, resulting in higher titers, higher capsid percentages, and / or higher infectivity.

[0052] Fig. 9A A suitable workflow for the production of AAV vectors using suspension HEK 293 cells as hosts is shown. Left: Cell inoculation, transfection, and harvesting of AAV particles from lysed cells. Right: Product quality assessment: capsid quantification, viral titer (or capsid concentration), full capsid fraction, and transduction function titer. 5. Demonstration of high-level virus production from confluent cells

[0053] Cell fusion of HEK 293 cells was performed multiple times using PEG as a fusing agent to form self-hybrids (multiple cells from one cell line).

[0054] To package and produce adeno-associated virus, serotype 2 (AAV2), unengineered and engineered HEK 293F were transfected with a helper plasmid, viral plasmin containing AAV Rep and Cap proteins, and a transfer plasmid expressing NeonGreen fluorescent protein under the control of the constitutive cytomegalovirus (CMV) early promoter, flanked by AAV2 inverted terminal repeats (ITRs) and another plasmid mi342 expressing microRNA under the control of the ubiquitous CMV promoter. Linear polyethyleneimine (PEI) was used for transfection. After transfection, crude virus was extracted from cell lysates and recovered by centrifugation. Quantitative PCR was used to measure the number of viral copies produced by unengineered and engineered HEK 293 cell lines.

[0055] Fig. 9B The number of viral particles that can be produced from fused cells is shown. The viral genomes of packaged AAV2 were measured by quantitative PCR after DNase I treatment. Quantification was performed using primers targeting the NeonGreen gene, and the vg / ml of the samples was determined using a commercial AAV2 reference virus from Vigene Biosciences (whose vg / ml is known). The engineered HEK 293F produced a 2.5-fold increase in viral genomes compared to the unengineered HEK 293F cell line. 6. Detailed plan

[0056] By way of example (without implying any limitation to the claimed invention and its equivalents), a production cell line of an AAV vector was obtained according to the following scheme:

[0057] Step 1: Generation of hybrids. Using a starting cell population of HEK 293 cells, cell hybrids were prepared by using polyethylene glycol as a fusion agent combined with gentle centrifugation to promote cell contact. Hybrids were cloned. Each clone was divided into aliquots and sampled for transfection testing.

[0058] Step 2: Transfection. Using a chemical-based approach, hybrid cell clones in suspension are transfected with lipopolymers that complex with negatively charged DNA to form lipopolyplexes through electrostatic interactions. Three plasmid vectors are used for transfection: 1) a transfer vector that expresses a fluorescent protein under the control of a ubiquitous CMV promoter cassette flanked by AAV inverted terminal repeats; 2) a helper vector cassette that expresses the adenovirus E4 gene for AAV DNA replication, the adenovirus E2a gene, and the adenovirus VA RNA (virus-associated RNA) gene to enhance AAV mRNA stability and promote AAV capsid transcription; and 3) a packaging vector that expresses the Rep and Cap proteins for specific serotypes (AAV1, AAV2, and AAV5). Cells were harvested 72 hours after transfection, lysed, and the AAV yield was determined.

[0059] Step 3: Determine the productivity of the cloned hybrids. AAV genome copy number was measured by real-time quantitative PCR. Cell lysates were treated with DNase I to remove non-viral host genomic DNA. Real-time quantitative PCR was performed by fluorescence detection to determine the viral genome copy number. DNA primers bind to the coding region of the fluorescent reporter gene within the transfer vector transfected in the assembled AAV, and the copy number is detected using fluorescence (the method used in the previous figure).

[0060] Step 4: Determine AAV serotype-specific capsid. Biolayer interferometry (BLI) is an optical biosensing technique that allows real-time analysis of biomolecular interactions without the need for fluorescent labels. The white light interference pattern or phase shift caused by the binding of the analyte sample to the immobilized ligand on the biosensor probe is used to quantify the amount of AAV virus in the unknown sample. A small biosensor that specifically binds to AAV capsid proteins of multiple serotypes (AAV1, AAV2, AAV5). For each serotype (AAV1, AAV2, and AAV5), a standard curve of commercial AAV reference standards (known concentrations measured by other validated methods) is used to back-calculate the concentration of the AAV serotype in the unknown sample.

[0061] Step 5: Measure the ratio of full capsids to empty capsids. The ratio of full capsids to empty capsids in unknown samples can be measured by biolayer interferometry. First, the concentration of AAV serotypes of unknown samples is measured as described above. For each serotype to be measured, a standardized concentration of AAV capsids is 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 conjugated to the SSB protein, which specifically binds to the ssDNA. For each serotype (AAV1, AAV2, and AAV5), a standard curve of a commercial AAV reference standard (the ratio of known full capsids to empty capsids measured by other validated methods) is used to reverse calculate the AAV serotype ratio in the unknown sample.

[0062] Step 6: Measuring functional titers. To measure functional titers, undiluted samples of AAV1, AAV2, and AAV5 generated using clonal hybrids were infected at certain dilutions and added to a fixed population of uninfected HEK 293 cells. Functional titers were measured in transducing units (TU) per milliliter (mL). Infectivity was determined by quantifying the percentage of fluorescent positive cells by flow cytometry.

[0063] Step 7: Expand high-producing clones. Amplify original aliquots corresponding to samples showing high levels of capsid production and functional titers to establish producer cell lines for transduction and expression of other types of viral vectors and particles.

[0064] Fig. 10A , 10B and 10C show that cells fused, cloned, and sampled according to this figure have higher productivity. Host cells were transiently transfected with (1) a transfer plasmid expressing a fluorescent reporter gene; (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 AAV1, AAV2, or AAV5 capsids. Cumulative capsid productivity of AAV1, AAV2, AAV5 ( Fig. 10A ), cell specific productivity (VP / cell) ( Fig. 10B ) and percentage of full capsid ( Fig. 10C The engineered clone (17-2) increased 3-fold, 9-fold, and 2.5-fold, respectively, compared to the parental host.

[0065] Fig.11A and 11BIt is shown that cells fused, cloned and sampled according to this figure have higher functional titers. Concentrated packaged AAV1, AAV2 and AAV5 viruses were infected with HEK 293T cells at different multiplicities of infection (MOI), and the percentage of infected cells was determined by gating on fluorescent positive cells using flow cytometry. Fig. 10A : The average functional titer was calculated at three different virus concentrations (dilution range from 1:25 to 1:300). Fig. 10B : Cellular infectivity was measured by the percentage of cells showing GFP fluorescence (AAV transfer vector transgene) at 3 different multiplicities of infection (MOI), ranging from 500 to 50,000 (serotype dependent).

[0066] Fig.12 The increase in the proportion of capsids containing nucleic acid payloads achieved in this figure is shown. Host cells were transiently transfected with transfer plasmids expressing fluorescent reporter genes, packaging plasmids expressing AAV1 or AAV2 specific Rep and Cap proteins, and helper plasmids. The full-to-empty ratio of AAV1 and AAV2 of lysed cells was measured by BLI. The engineered HEK 293T 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-to-empty ratio. 7. Preparation of Cell Hybrids

[0067] Individual high-producing cells can be selected from any population of cells that is heterogeneous in this respect, as described in the following section. Many single cell lines (e.g., CHO and HEK-293 cells) are sufficiently diverse at the outset in terms of the genetic content and intracellular apparatus of the proliferating cell population that they can be sorted and selected for high-producing cells directly from standard cultures.

[0068] Alternatively, to increase final product yield or enhance the sorting process, the user can prepare cells for sorting by employing a combination of one or more techniques that will enhance the heterogeneity of virus production levels within a population of cells, or generally increase virus production levels across a population of cells or a subpopulation thereof. Suitable techniques are those that alter the genome of the cell, for example, shuffling the genome and increasing the copy number of intracellular machinery that facilitates viral production or assembly. Altering or shuffling the genome in this manner may generate a number of genetic variants with one or more of a variety of different properties, including virus production levels and growth rates.

[0069] The technical part of the present disclosure is based on the following disclosure: cells that are suitable for virus production can reach higher production levels by merging with other cells. Without limiting the practice of the present invention, it is assumed that two cells are fused together to have partial additivity in the composition, genetics or genetic control of the cells involved in virus production. If the improved characteristics can be reproduced, it is beneficial. Therefore, after cell fusion, they usually go through multiple rounds of cultivation and selection to obtain the phenotypic characteristics of the target. The resulting cells may be aneuploid, or otherwise retain all or part of the genome of the parental cells encoding the cell components involved in virus production.

[0070] Model cells suitable for fusion are cell lines that have been used for industrial virus production, such as Chinese hamster ovary syndrome (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, "cell line" is a cell population that can be continuously, extensively or indefinitely propagated in tissue culture. The starting cell line is usually heterogeneous in one or more phenotypic characteristics, which are related to the amount of the transgenic gene product that the cell will produce. When cultivating, the production cell line obtained according to the present disclosure can produce heterogeneous, substantially homogeneous or cloned offspring.

[0071] Cell fusion is performed by obtaining a cell mixture of cells to be fused: a plurality of cells from one cell line or a plurality of 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 hybrid formation, by performing electrofusion, by combining with a fusion virus (e.g., Sendai virus), by contacting the cells (e.g., by gentle centrifugation), by treating with a fusogenic agent (e.g., polyethylene glycol (PEG)), or using any effective combination thereof.

[0072] For purposes of this disclosure, cells made by fusing two or more cells together may be referred to as autotypic hybrids (cells from the same cell line fused together), isotypic hybrids (cells with the same genotype), allotypic hybrids (cells from different individuals of the same species with different genotypes), and xenotypic hybrids (cells from different species). Autotypic hybrids are typically formed using a cell population that is substantially (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 with potentially complementary properties. The present disclosure also includes fusing one or more cell populations isolated or obtained from a primary source with itself or with an established or cloned cell line.

[0073] Any suitable technology can be used to fuse cells into hybrids. For example, cells can be cultured under the presence of a fusogenic agent and / or under the culture conditions that promote the formation of hybrids, or can be forced to contact, for example, by gentle centrifugation, optionally combined with a fusogenic agent such as polyethylene glycol (PEG). Usually, fused cells are obtained by fusing two cells together, although the fusion of three or more cells is also possible. It is recognized that the fusion of two different cell populations will produce mixed cell products (homotype, allotype or heterogeneous hybrid, depending on the parental cell line) and self-type hybrids. If desired, fluorescently labeled or surface-bound antibodies can be used to separate self-type or homotype hybrids from allotype or heterogeneous hybrids, and the antibody has specificity to the part expressed on a cell line in the mixture, but is not specific to another cell line.

[0074] Unless otherwise expressly stated, all such combinations are within the scope of the present invention. It may be beneficial to repeat cell fusions in hybrid populations to further enhance the effect and / or cross-hybridize with other cell lines to impart additional beneficial properties to the final cell line. Thus, the fusion and selection steps may be iteratively performed twice, three times, four times or more. 8. Selection of high-producing cell lines and preferred phenotypic characteristics

[0075] The present disclosure provides a variety of methods for identifying and selecting cell hybrids with the ability to produce high-yield cell lines. Cells can be transfected with reporter genes (e.g., genes encoding fluorescent products such as green fluorescent protein) and genes encoding viral capsids for testing purposes. High-yield cells can be selected based on the viral capsids produced and / or the encapsulated promoter gene products.

[0076] Alternatively or additionally, cell hybrids can be selected for characteristic phenotypes that are often associated with high levels of protein and / or viral product. A valuable insight behind this technology is that by selecting cells from a mixed cell population that have optimal levels of subcellular machinery or biochemistry compared to other hybrids or parental cells in the starting mixture, the yield of biologics can be increased, thereby supporting increased viral yields.

[0077] Such phenotypic characteristics include the relative density of subcellular organelles, particularly those involved in secretion of proteins or viral particles from the host cell, and the relative levels or concentrations of enzymes that help complete or assemble the virus. These include mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, and nucleoli. Such phenotypic characteristics also include various aspects of the cell's cytosol or cellular contents, such as reactive oxygen species, redox-carrying molecules, and pH.

[0078] Depending on the viral system to be optimized, it may be preferred to have 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 an appropriate vital dye and divided into low (bottom 10% or bottom 5% to 25%), medium (middle 30% to 70%), or high (top 10% or top 5% to 25%) aliquots based on each feature of each cell using a cell sorter or other method. Hybrid cells falling within these ranges can be recovered and aliquoted or cloned and then tested for viral yield and effective titer. The range determined to be advantageous can then be used as an additional standard for finding other high-yield aliquots or clones of related viral serotypes or systems.

[0079] The makers of the present invention have discovered that fused cells sorted for higher mitochondrial content and higher reactive oxygen species (ROS) levels can be used to produce production cells that produce viral capsids, wherein the proportion of capsids produced by the production cells that contain the intended drug payload (e.g., polynucleotides for gene therapy or vaccination) can be twice as high. 9. Selecting the Cellular Content of Mitochondria

[0080] Many viral proteins localize to mitochondria. The content and function of mitochondria are used as the basis for sorting or selection using vital dyes without damaging the cells. Such dyes are commercially available, for example, from the following companies: Invitrogen and Sigma Aldrich. Examples of vital dyes for mitochondria include: MitoTracker Green FM; MitoTracker Orange CMTMRos; MitoTracker Red CMXRos; MitoTracker Red FM; MitoTracker Deep Red FM; BioTracker 488Green Mitochondria dye; BioTracker633Red Mitochondria dye; BioTracker405; and Blue Mitochondria.

[0081] Functional dyes for measuring the membrane or redox potential of mitochondria can also be used to sort or select cells with enhanced mitochondrial function. Mitochondrial potential is generated by complexes I, III, and IV and can be used as a reliable readout to assess mitochondrial function. Membrane depolarization shifts the fluorescent signal from one wavelength to another. These membrane potential dyes are available from the following companies: JC-1 Dye (Invitrogen T3168; Sigma CS0390); JC-9 Dye (Invitrogen D-22421); and C10 Dye (Sigma MAK160, MAK159).

[0082] Other features used to classify enhanced mitochondria include vital dyes for measuring mitochondrial calcium, superoxide production, and dyes selective for mitochondria. These include Rhod-2AM Reagent (Invitrogen R1245MP); and MitoSOX Red (Invitrogen M36008).

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

[0084] Following staining with any of these dyes, cells can be selected (e.g., by flow cytometry and sorting) whose staining levels are, on average, at least 1.2, 1.5, 2, or more times higher than that of the parental cell line, e.g., for mitochondrial or optically labeled gene products. 10. Select peroxisomes, cellular content of reactive oxygen species and pH

[0085] Peroxisomes are plastic subcellular organelles present in almost all eukaryotic organisms. They act as external sensors of the cell and as centers for reactive oxygen species (ROS), lipid and amino acid β-oxidation (reviewed in Smith and Aitchison, 2014). Peroxisomes can be labeled with expression-based markers, where fluorescent proteins are targeted to peroxisomes using peroxisome targeting sequences.

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

[0087] Following staining with any of these dyes, cells can be selected (e.g., by flow cytometry and sorting) for cells having an average staining level that is at least 1.2, 1.5, 2, or more times higher than that of the parental cell line, e.g., staining for a peroxisomal or optically labeled gene product.

[0088] The present disclosure demonstrates for the first time that increased cellular levels of ROS are associated with increased viral production. 2- ), hydrogen peroxide (H2O2), and hydroxyl radicals (HO·), which constitute free radical and non-radical oxygen species formed by partial reduction of oxygen. Cellular ROS are generated endogenously, such as during mitochondrial oxidative phosphorylation, and are associated with a variety of pathological diseases, such as cancer, neurodegenerative diseases, and aging.

[0089] Cellular reactive oxygen species can be measured using fluorescent probes, where upon oxidation, these agents exhibit strong fluorescence and remain localized within the cell. These dyes are commercially available and include the following: From ThermoFisher: Green; Orange; Deep Red; and H2DCFDA. From Abcam: DHE (Dihydroethidium) Assay Kit.

[0090] OxyBURST Green reagent, RedoxSensor RedCC-1 stain, and reduced calcein, ethidium, fluorescein, MitoTraker probes, and rhodamine can be used to determine intracellular redox levels. 9-amino-6-chloro-2-methoxyacridine (ACME), BCECF indicators, dextran conjugates, fluorescein and fluorescein derivatives, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), LysoSensor probes, Oregon Green dye, pHrodo indicators, SNARF indicators, and thiol-reactive pH indicators can be used to determine intracellular pH. 11. Select the level of other intracellular organelles

[0091] Alternatively or additionally, high producing cells may be identified or selected based on the intracellular content of other organelles.

[0092] Select the endoplasmic reticulum (ER) and / or the Golgi apparatus. These organelles play a central role in protein production. One or both can be measured and used as the basis for sorting or selection using vital dyes without damaging the cells, and cells can be selected based on the amount of dye associated.

[0093] Such dyes are commercially available, for example, from Molecular Probes, Inc. Examples of ER vital dyes include: ER-Tracker TM Blue-White DPX(E12353);ER Tracker TMGreen(Glyburide FL)(E34251); ER-Tracker TM Red(Glyburide TR)(34250); DiOC6(D273); and DiOC5(D272). Golgi vital dyes include NBD C6-6-ceramide(N1154); NBD C6-sphingomyelin; FL C5-ceramide (D3521); and TR Ceramide (D7540).

[0094] Alternatively or in addition, users can test expression-based labeling systems that introduce fluorescent proteins that target the ER or Golgi. These are fusion proteins that contain a portion that expresses the optical label fused to a protein sequence that is targeted to or processed by the organelle to be labeled. Examples include the following: From Invitrogen: CellLight TM ER-GFP(C10590);CellLight TM ER-GFP(C10591);CellLight TM Golgi-GFP(C10592);CellLight TM Golgi-GFP (C10593). From 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] Following staining with any of these dyes, cells can be selected (e.g., by flow cytometry and sorting) for cells having an average staining level that is at least 1.2, 1.5, 2, or more times higher than that of the parental cell line, e.g., for a gene product of the ER, Golgi, or an optical marker.

[0096] Select for high cellular content of nucleoli. The nucleolus is the largest subnuclear organelle in the cell, where ribosomal RNA is assembled and transported to the cytoplasm and supports the protein translation machinery. The nucleolus in the cell can be labeled with vital dyes. Fluorescent probes targeting ribosomal RNA can also be used to selectively stain this subcellular organelle. Examples include the following: Nucleolar Staining Kit (Abcam, ab139475); NUCLEOLAR-ID Green Detection Kit (Enzo Life Sciences, 51009-500); and SYTO RNA Select Green (Invitrogen, S32703).

[0097] Following staining with any of these dyes, cells can be selected (e.g., by flow cytometry and sorting) for average staining levels that are at least 1.2, 1.5, 2, or more than 2-fold higher, e.g., for the nucleolus or an optically-marked gene product. 12. Characterization of High-yielding Cell Lines

[0098] Cell hybrids optimized for production of viral vectors and particles can be characterized by one or more criteria in any combination.

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

[0100] The above materials can also be used to characterize producer cells based on cellular phenotype (e.g., intracellular content of mitochondria, peroxisomes, reactive oxygen species (ROS), endoplasmic reticulum, Golgi apparatus, nucleoli, etc.). 13. Determine the production capacity and characteristics of producer cells

[0101] After screening, cell lines or mixed cell populations that produce high levels of virus can be compared to the parental or original cell line and characterized by one or more different parameters. For example, the selected cells can have: (1) a genome that is more aneuploid than the starting cell, containing part or all of the genome of two or more parental cell lines (which may be the same or different), (2) a higher concentration of mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, reactive oxygen species, or other phenotypic characteristics compared to any or all of the parental cell lines (e.g., 2 to 5 times or 4 to 8 times higher, or 2, 4, or 8 times higher), (3) a significantly higher ability to produce virus levels per cell or per liter of culture medium than the parental cell line (e.g., 2 to 5 times or 4 to 8 times higher, or 2, 4, or 8 times higher), (4) a significantly higher ability to produce virus levels per cell or per liter of culture medium than the parental cell line (e.g., 2 to 5 times or 4 to 8 times higher, or 2, 4, or 8 times higher, or 3) a significantly higher ability to produce virus levels per cell or per liter of culture medium than the parental cell line (e.g., 2 to 5 times or 4 to 8 times higher, or 3, or 10 to 50 grams of virus per liter of culture medium; or (6) are capable of producing viral vectors or particles having a higher proportion of capsids carrying payload (e.g., 50%, 80%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 2000%, 5000%, or 20,000 capsids per cell).

[0102] To make such comparisons, the producer cell line can be compared to a standardized population of the original cell line that is either kept on hand as part of the same system or obtained from a reference source. For example, CHO-derived producer cells can be compared to the American Type Culture Collection. The disclosure includes a system for high-level production of a virus-based drug, the system comprising a starting cell line and a production cell line derived therefrom, the production cell line having a relatively high mitochondrial density and / or reactive oxygen species (ROS) density per cell, for example, as determined using one or more of the above-described activity dyes. 14. Genetically engineer producer cells to synthesize and produce viral elements

[0103] To generate cell lines expressing viral gene products, production cells or their precursors can be transfected with a single gene encoding a viral element (e.g., a protein and a nucleic acid). More commonly, multiple vectors are used to introduce viral elements into host cells.

[0104] The expression of the gene cassette can be controlled by a combination of mammalian promoters: ubiquitous endogenous viral promoters (non-ubiquitous, such as 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 placed in the forward or reverse orientation relative to the promoter. The gene or genes can be flanked by recombination sites (FRT and its variants; and / or lox and its variants). These recombination site variants include: loxP, lox511, lox2272, FRT or mFRT71. A site-specific recombinase (such as Cre or Flippase) is expressed in the same cell to allow site-specific recombination and change the orientation of the gene from reverse to forward.

[0105] For example, recombinase can be expressed by transient transfection, wherein the gene encoding the recombinase is controlled by a ubiquitous or inducible mammalian promoter. In another embodiment, purified recombinase protein or mRNA can be transfected into cells. In another embodiment, recombinase can be delivered using adenovirus, slow virus, AAV, Moloney murine leukemia virus (MMLV), mouse stem cell virus (MSCV), vesicular stomatitis virus (VSV) or herpes simplex virus (HSV). By using polycistronic elements (such as T2A, P2A, E2A, F2A, IRES and IRES2 elements), multiple genes can be expressed by the same promoter. Inducible promoters cause the expression of genes or gene cassettes when adding stimulants, and the stimulants can be chemical stimulants (such as doxycycline, tetracycline, cumate, recombinase (such as Cre or Flippase)) or physical stimulants (such as blue light). The level of target protein production can be determined during the process of inserting the gene expression cassette using transient transfection methods.

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

[0107] Adenovirus, adeno-associated virus (AAV), and lentivirus can be produced by transiently transfecting one or more combinations of the following vectors into a cell line: a helper vector, a packaging vector, an envelope vector, and / or a transfer vector. The gene cassettes of the helper vector, packaging vector, envelope vector, and transfer vector vary depending on the type of virus produced. The helper vector can express the E2A and E4 genes as well as the VA RNA of adenovirus and AAV. In another embodiment, the packaging vector expresses the Rep and Cap genes of adenovirus and AAV, while a different packaging vector expresses Gag, Pol, Rev, and its response elements produced by the lentivirus.

[0108] Alternatively, the Rep or envelope gene can be expressed in an inducible manner, wherein the Rep gene cassette is divided into two fragments (5′ and 3′ fragments) and the two fragments are connected by a termination cassette containing transcription termination and polyadenylation elements flanked by two homologous recombination sites located in cis. In another preferred embodiment, the E2, E4 and VA cassettes are placed under the control of an inducible promoter in the opposite direction to the promoter. Activation of EEV is achieved by delivering Cre and Doxycyline. In a preferred embodiment, loxP and lox511 are used for recombination. Other heterologous recombination sites can be used: lox2272, FRT and mFRT71. In the case of using any combination of FRT and mFRT71 recombination sites, Flippase must also be delivered to the cell. Envelope vectors are only used for the production of lentiviruses, not adenoviruses or AAVs. The transfer vector backbone containing the gene of interest is unique to the virus type. The lentiviral transfer vector backbone consists of the following components: 5′ and 3′ LTRs, the Psi packaging signal, and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). The adenoviral and AAV transfer vector backbones contain an inverted terminal repeat-flanked promoter, the gene of interest, and the WPRE.

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

[0110] Lentiviruses can be produced by transfecting packaging, envelope and transfer vectors into a parental host cell line. In another embodiment, lentiviruses are produced by transfecting a transfer vector (expressing the gene of interest) into a cell line containing stably integrated helper and packaging elements. In another embodiment, the virus is produced in a cell line with stably integrated helper, packaging and transfer vectors.

[0111] Transfection can be done using liposome-based reagents (e.g., Lipofectamine TM 3000, Expifectamine293, FuGENE TM HD, X-Fect nanoparticle polymers, Trans-IT Pro reagent, Trans-IT VirusGen, polyethyleneimine), calcium phosphate, electroporation, or infection with adenoviral, retroviral, or lentiviral vectors.

[0112] After transfection, the cells are tested for the yield of the target virus for packaging: for example, by enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), or biolayer interferometry (BLI). Select cells or clones with increased yields of the desired virus. The goal can be a 1.5, 2, 4, 8, 12, 16, 20, or 100-fold increase in viral yield over the parental cell line; and / or production at a level greater than 1012 viral genomes / ml or capsid / ml for AAV; and / or greater than 108 infectious units (IFU) per milliliter of culture fluid under typical manufacturing conditions. Other desired properties of the target virus, such as the ratio of full capsids to empty capsids and functional titers, can also be tested.

[0113] In principle, transfection can be carried out before, during or after one or more fusions and the circulation of selecting other characteristics.For example, before transfection with packaging, auxiliary and transfer vector containing target gene, fusion and selection can be carried out, thereby set up the parental cell line of high-level virus production that is suitable for user selection.Or, can be transfected in the original parental cell line containing target gene, and be used for tracking production level in subsequent fusion and sorting step, or provide another basis for this type of sorting.Or, can be carried out as an intermediate step, wherein cell has experienced the fusion of one or more circulations and the selection for some other characteristics, such as ER, Golgi body, mitochondria, peroxisome, nucleolus or other proteins (mentioned in the front of the present disclosure).Gained hybrid is transfected to express target virus, then further merges and selects circulation to express target virus and / or other characteristics mentioned above in the present disclosure.

[0114] Another option is to develop a cell line using a reporter gene as a proxy for the viral payload that will ultimately be manufactured: for example, secreted alkaline phosphatase, secreted luciferase, a fluorescent viral payload (such as a red fluorescent viral payload or a green fluorescent viral payload). Again, transfection can be performed before, during, or after multiple cycles of fusion and selection, optionally using the expression level of the marker as a selection criterion in one or more cycles. This creates a parental cell line that is optimized for expression of the marker viral payload, with the expectation that the beneficial characteristics of the cell line will be retained after further genetic engineering to produce a commercially valuable biological product.

[0115] Finally, once a cell line with a desired level of expression of a tagged viral payload is developed, the tag is replaced with a target viral payload. The techniques listed above can be used to randomly transfect into the genome again and limit the expression of the reporter gene. Alternatively, targeted integration techniques can be used to replace the gene of the reporter gene with a gene encoding a target viral payload. Such techniques include, for example, CRISPR / Cas viral payloads, CRISPR / Cas associated transposases (CAST), recombinase cassette exchange (RMCE), zinc finger recombinases (ZFRs), or transcription activator-like effector nucleases (TALENs). In this way, the target gene is inserted into the genome of a cell from a production cell line or mixture, and the pre-selected position allows or supports high-level transcription compared to other positions in the genome. 15. How to use transposase to stably transfect producer cells

[0116] Transposons are DNA sequences that can move one location in the DNA by (1) copy and paste or (2) cut and paste mechanisms. They have recently emerged as a promising toolkit for gene amplification in molecular biology, where up to 50 copies can be introduced into the genome. In contrast to transposons, conventional transfection techniques using chemical-based methods or electrical currents can only integrate a single or very few copies of a transgene into the host genome.

[0117] For example, the class II transposon "Sleeping Beauty" can be used to stably introduce multiple copies of viral genes into cells. By predictably introducing a desired number of copies of a specific viral gene into cells, the benefits of maximizing viral yield can be achieved. The optimal ratio of viral proteins is critical for the correct packaging of lentiviruses, adenoviruses, and AAVs, and can vary depending on the type of virus. The idea of ​​using class II transposons (Sleeping Beauty and PiggyBac) to amplify the number of viral gene copies integrated into cells can also be applied to class I transposons and CRISPR transposons (CASTs; Mougaikos and Beisel, 2021).

[0118] Multi-component molecular systems, such as the SunTag system, can be used to amplify gene expression of viral genes in cell lines without the use of the Sleeping Beauty transposase system. Global transcription factors, such as Tat or p300, are tagged with multiple SunTag scaffolds containing multiple copies of the GCN4 epitope. The homologous scFv fragments are fused to a heterotypic fusion protein consisting of transcriptional activation domains: p53, VP64, p65, and Rta (SSPVP), see Figure 3. When these components are co-expressed in cells, these components are called super transcription activation complexes (STACs), which can lead to gene expression amplification of viral genes in a synergistic manner. Compared to amplifying gene copies using transposase systems, the use of such systems can achieve the benefits of gene expression amplification of cells with a low number of integrated gene copies. This will allow amplification beyond the normal levels of endogenous biological systems. 16. Virus strains suitable for treatment

[0119] After appropriate modification, the technology of the present disclosure can be implemented in any virus strain selected by the user. Table 1 provides some examples of viral genetic elements that can be transfected into the production cell lines of the present disclosure to prepare lentiviral, adenoviral and AAV vectors. 17. Promoters for expressing viral gene elements stably transfected into production cell lines

[0120] If the producer cell line is stably transfected to integrate the viral transgene into the genome of the cell, it is sometimes helpful to regulate such cells if the promoter used to drive expression of the viral gene in the producer cells is inducible.

[0121] One embodiment is benzoic acid p-isopropyl ester (cumate) inducible promoter (CymR), which is a repressor that binds to benzoic acid p-isopropyl ester operator sequence (CuO) in the absence of benzoic acid p-isopropyl ester. In the presence of benzoic acid p-isopropyl ester, Cumate binds to CymR, thereby activating genes downstream of CuO. U.S. Patent Nos. 8,728,759 and 7,745,592B2. Tetracycline-response element (TRE) is also applicable, which can be induced using Doxycyline or tetracycline. Light-inducible promoters can also be used, such as blue light-inducible promoters from GenTarget, Inc. 18. Methods for quantifying virus production levels in cell hybrids

[0122] Real-time quantitative PCR measures viral transcription, viral genome concentration (vg / ml). Each viral particle typically contains one viral genome. The virus is treated with DNase I to remove any host genomic DNA. Primers that bind to the targeted region in the transfer vector are used and the amplicon is detected by a probe-based approach or SYBR Green bound to the amplicon.

[0123] Total capsid AAV particles were measured using indirect ELISA and biolayer interferometry (BLI). These measurements utilized antibodies against the abundant capsid proteins present in AAV serotypes. Samples were captured by capsid antibodies and chemiluminescent detection was performed using biotinylated capsid antibodies and streptavidin conjugated to HRP.

[0124] For lentiviruses, infectious particles can be measured by indirect or sandwich ELISA using antibodies against p24. Anti-p24 is used to capture the sample and chemiluminescent detection is performed using biotinylated anti-p24 and streptavidin conjugated to HRP.

[0125] The functional titer or infectious titer of a virus is the concentration of viral particles that can transduce cells. Functional titer can be measured by cell transduction using fluorescent or chemiluminescent proteins as reporter genes. Cell lines are infected or transduced with packaged viruses at a specific multiplicity of infection (MOI). The percentage (%) of cells expressing the reporter gene is quantified and correlated with the number of viral particles used to transduce cells. 19. Drug payloads and therapeutic applications

[0126] The viral vectors and particles produced according to the present disclosure can be used to deliver various drug payloads to human subjects in need. Suitable are various types of proteins and nucleic acids, or combinations thereof. Treatment is accomplished by administering to a subject an amount of the vector or particle that is effective to achieve one or more clinical purposes.

[0127] The technology disclosed herein is advantageous for delivering nucleic acids, proteins or mixtures thereof to induce an immune response of the opposite sex. 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 encodes one or more additional proteins that can serve as adjuvants or stimulants to enhance immunogenicity. The target can be an infectious pathogen, such as a pathogenic virus, bacterium or protozoa. Alternatively, the target can be a cancer cell, in which case the encoded epitope is an epitope expressed by a cancer cell that is specific to a cancer or tissue type.

[0128] For example, the technology disclosed herein can be used to prepare compositions that induce responses to the SARS CoV 2 virus for the prevention or treatment of COVID 19. Representative immunogenic epitopes can be taken from one or more of the four SARS-CoV-2 structural proteins: 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 were recently discussed in (F. Heinz & K. Stiasny, NPJ Vaccines (2021) 6: 104).

[0129] The technology disclosed herein can also be used for the purpose of gene therapy: for example, delivering nucleic acids encoding gene products that are missing or defective in the subject being treated, or targeting pathogenic cells in the subject, particularly cancer cells. Therapeutic purposes include, but are not limited to, expressing therapeutic proteins encoded in nucleic acids (e.g., cytokines or anticancer agents), expressing essential proteins that the subject cannot produce on its own, or delivering gene editing systems (e.g., CRISPR / Cas9 or guide RNA). Other possible therapeutic payloads may include DNA antisense oligonucleotides, DNA aptamers; microRNAs, short interfering RNAs, ribozymes, RNA baits, and circular RNAs that specifically increase or decrease the expression of specific endogenous genes in subjects or infectious sources. (K. Sridharanet a1., BrJ Clin Pharmacol. 2016 Sep; 82(3): 659672).

[0130] Exemplary payloads for gene therapy are shown in Table 3. In the examples shown, the nucleic acids encode therapeutic antibodies (for passive immunization), anticancer drugs such as cytokines and chemokines (for cancer treatment), and natural human proteins (to promote the synthesis of essential factors that the subject may lack, such as in the case of genetic diseases). Tables 2 and 3 are adapted from (X. Hou et al., Nat Rev Materials 2021, 10: 117). 20. Drugs and commercial products

[0131] Preparation and formulation of medicaments for use according to the present disclosure can be combined with standard techniques, for example, as described in the latest edition of Remington: The Science and Practice of Pharmacy. The formulation is generally optimized for systemic administration (intramuscular or subcutaneous administration) or oral or nasal administration (e.g., to stimulate the mucosal immune system).

[0132] The formulations of viral vectors and particles can be provided in the form of one or more unit doses (combined or separate), each unit dose containing an amount of a drug payload that is effective in treating a selected disease, infection, or clinical condition. The commercial product may include a device (e.g., a syringe) for administering the agent or composition into or around a target tissue of a subject in need thereof. The product may also include or be accompanied by an information package insert describing the use and concomitant benefits of the vector or particle in treating the disease for which it is indicated and approved. Example Example 1: Protein production by cell hybridization

[0133] In this example, to maximize protein production, CHO cells were fused and sorted to extract cells with high endoplasmic reticulum (ER) content.

[0134] CHO-K1 cells were exposed to the PEG-assisted fusion procedure. The cells were allowed to recover for one week and then the procedure was repeated three times. After recovery from the third fusion, the cells were stained with a live ER tracking dye (ER Tracker TM Green(Glyburide FL); Invitrogen, E34251) were used to stain the cells and the FACSAriaII TM Cells were sorted using a cell sorter (BD Biosciences). The 10% of the viable cell population that showed the highest amount of ER Tracker dye staining was collected. After two weeks of recovery in culture, cells were exposed to terminal confluence, stained with ER Tracker dye, and analyzed using LSRII TM Analysis was performed by flow cytometry (BD Biosciences).

[0135] To measure protein production in fusion cells and parental CHO populations, cells were transfected to express secreted alkaline phosphatase (SEAP). Transfection was performed as follows: 1. Centrifuge 106 cells. 2. Discard the supernatant 3. In 100μL Cell Line Nucleofector TM Resuspend in Solution T 4. Add 2 μg SEAP expression plasmid 5. Transfer to the Electroporation Cuvette 6. Use Amaxa TM Electroporation with Nucleofector II and preset program U-023 7. Add 0.5 ml of growth medium 8. Transfer cells to a 6-well plate containing 1 mL of growth medium per well.

[0136] Figure 7 Results are shown (specific productivity of secreted alkaline phosphatase). Expression of the marker protein (SEAP) in fusion cells showed more than 4-fold improvement. Example 2. Generation of AAV packaging cell lines containing Rep-Cap and EEV gene cassettes using the PiggyBac and Sleeping Beauty transposase systems. 2.1 Generation of a cell line containing Rep-Cap (fHEK-RC)

[0137] The vector containing the Rep-Cap gene cassette ( Figure 2 A) Transfection into confluent HEK 293F (fHEK) cells. Vector DNA and the PiggyBac transposase vector were added to the cell suspension, followed by electroporation using a single pulse exponential decay. After transfection, selection medium containing a selective concentration of blasticidin was added to the culture medium, and surviving cells containing stably integrated Rep-Cap were transfected with the EEV cassette using the Sleeping Beauty Transposase System ( Figure 2 B).

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

[0139] Figure 3B: Inducible cassettes for activation of E2A, E4 and VA RNAs, abbreviated EEV and Cre recombinase. The inducible cassette is flanked (left and right (abbreviated as LTRs and RTRs, respectively)) by Sleeping Beauty transposon inverted terminal repeats. EEV is in reverse orientation and flanked by heterologous LoxP sites. Upon activation by Doxycyline, cis-recombination will result in EEV in the correct forward orientation, leading to their expression in a dose-dependent manner. Cre recombinase is controlled by Doxycyline via the TRE promoter (see below). Dox binds to the reverse tetracycline-controlled transcription activator (rtTA) to form the Dox-rtTA complex. The Dox-rtTA complex binds to the Tetracycline-responsive element (TRE) to activate downstream target genes. The repressor, the reverse tetracycline-controlled trans-silencer (rtTS), has two functions: (1) minimizes leaky expression from the TRE promoter; and (2) enhances rtTA activity in the presence of Doxycyline as a co-activator. Other abbreviations: Cbh, CMV and chicken β-actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 poly A tail. 2.2 Generation of cell lines containing Rep-Cap and EEV (fHEK-AAV)

[0140] Contains E2A, E4 and VA gene / RNA cassette (see Figure 2 B) and Sleeping Beauty Transposase (SBT) vectors were sequentially transfected into fHEK-RC by electroporation using a Bio-Rad Gene Pulser using a single pulse exponential decay. The transfected cells were grown in a selective medium containing blasticidin and hygromycin, and the surviving cells contained stably integrated Rep-Cap and EEV cassettes. Example 3. Generation of an AAV packaging cell line containing Rep-Cap and EEV cassettes (fHEK SAAV)

[0141] The vector containing the EEV and STAC cassettes (Figure 3) was transfected into fHEK-RC by electroporation using a Bio-Rad Gene Pulser with a single pulse exponential decay. The transfected cells were grown in a selective medium containing blasticidin and hygromycin, and the surviving cells stably integrated the Rep-Cap, EEV and STAC gene cassettes.

[0142] Figure 3C: Inducible STAC cassette for amplification of coordinated activation of E2A, E4 and VA RNA (EEV). The wings of the inducible cassette (left and right sides (abbreviated as LTRs and RTRs, respectively)) are connected by PiggyBac transposon inverted terminal repeats. Transcriptional coactivators and histone acetyltransferases (HATs), p300 and HIV-1Tat transcriptional activators are all linked to multiple GCN4 peptides (indicated by black rectangles). Both fusion proteins are expressed under the control of the CMV promoter. The hybrid scaffold SSPVP consists of a scFv fragment bound to the GCN4 peptide and the transcriptional activation domains of p53, VP64 and p65, and is used to recruit additional transcription factors, HATs and coactivators to the basal transcriptional machinery at the promoter.

[0143] Inducible cassette for activating E2A, E4 and VA RNA, abbreviated as EEV and Cre recombinase. EEV is in the reverse orientation and is flanked by heterologous LoxP sites. Upon activation of Doxycycline, cis-recombination will result in EEV being in the correct forward orientation, leading to their expression in a dose-dependent manner. Cre recombinase is controlled by 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-responsive element (TRE) to activate downstream target genes, which are only in the correct orientation upon Cre recombination events (e.g., EEV). The inhibitor reverse tetracycline-controlled transsilencer (rtTS) has two functions: (1) minimizes leaky expression from the TRE promoter; and (2) enhances rtTA activity in the presence of Doxycyline as a co-activator. Other abbreviations: Cbh, CMV and chicken β-actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 poly A tail. Example 4. Generation of lentiviral packaging cell line (fHEK-LV).

[0144] The vector containing the Gag, Pol, Tat and Rev (GPTR) gene cassette and the Bsd selection marker (see Figure 4A) was transfected into confluent HEK 293F (fHEK) cells by electroporation using the Bio-Rad Gene Pulser. The vector DNA and the PiggyBac transposase vector were added to the cell suspension and then electroporated using a single pulse exponential decay. The cells were grown in a medium containing a selective concentration of Blasticidin, and the surviving cells, referred to herein as fHEK-GPTR, were expanded and prepared for transfection with the VSV-G inducible gene cassette.

[0145] Figure 4A: Gag, Pol, Tat and Rev (GPTR) integration gene cassette. The GPTR gene cassette is flanked by the PiggyBac transposon left and right inverted terminal repeat elements (LTRs and RTRs, respectively). CAG is a hybrid promoter composed of CMV early enhancer elements, chicken β-actin promoter and rabbit β-globin gene splice acceptor. pA, SV40 poly A tail; Bsd, blasticidin resistance gene.

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

[0147] Figure 4B: Inducible cassette for activation of the envelope VSV-G gene and the gene encoding the Cre recombinase. The inducible cassette is flanked (left and right (abbreviated as LTRs and RTRs, respectively)) by the Sleeping Beauty transposon inverted terminal repeats. VSV-G is in the reverse orientation and is flanked by heterologous Lox (LoxP and Lox511) sites. The Cre recombinase is controlled by the TRE promoter (see below). Upon activation with poly-Doxycycline, Cre is expressed and cis-recombination results in VSV-G in the correct forward orientation, activating VSV-G 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-element (TRE) to activate downstream target genes. The inhibitor reverse tetracycline-controlled transsilencer (rtTS) has two functions: (1) to minimize leaky expression from the TRE promoter; (2) to enhance rtTA activity in the presence of Doxycyline as a co-activator. Other abbreviations: Cbh, CMV and chicken β-actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 poly A tail. Example 5. Generation of a lentiviral packaging cell line containing GPTR, inducible envelope and STAC gene cassettes (fHEK-SLV).

[0148] The vector containing VSV-G and STAC cassette ( Figure 5 ) were transfected into fHEK-GPTR. The transfected cells were grown in a selection medium containing blasticidin and hygromycin.

[0149] Figure 5 : An inducible STAC box for amplifying the VSV-G gene and the synergistic activation of the packaging genes Gag, Pol, Tat and Rev (GPTR, see Figure 4). The flanks of the inducible box (left and right sides (abbreviated as LTRs and RTRs, respectively)) are connected by the PiggyBac transposon inverted terminal repeat sequences. Transcription coactivators and histone acetyltransferases (HATs), p300 and HIV-1Tat transcription activators are all connected to multiple GCN4 peptides (represented by black rectangles). Both fusion proteins are expressed under the control of the CMV promoter. The hybrid scaffold, SSPVP, consists of a scFv fragment bound to the GCN4 peptide and the transcription activation domains of p53, VP64 and p65, which is used to recruit additional transcription factors, HATs and coactivators to the basal transcription apparatus at the promoter.

[0150] Inducible cassette for activation of the envelope VSV-G gene and the gene encoding the Cre recombinase. VSV-G is in the reverse orientation and flanked by heterologous Lox (LoxP and Lox511) sites. After activation with Doxycycline, Cre is expressed and cis-recombination will result in VSV-G in the correct forward orientation, activating VSV-G gene expression in a dose-dependent manner. After Doxycyline induction, Dox binds to the reverse tetracycline-controlled transcription activator (rtTA). The Dox-rtTA complex binds to the Tetracycline-element (TRE) to activate downstream target genes. The inhibitor, the reverse tetracycline-controlled trans-silencer (rtTS), has two functions: (1) to minimize leaky expression from the TRE promoter; (2) to enhance rtTA activity in the presence of Doxycyline as a co-activator. Other abbreviations: Cbh, CMV and chicken β-actin hybrid promoter; Hygro, hygromycin resistance gene; pA, SV40 poly A tail. 5.1 Other possible genetic modifications and potential benefits

[0151] Chromatin modifications can have both positive and negative effects on gene transcription. Actively transcribed genes are enriched for histone acetylation in the promoter and 5' region of the gene coding region. Among the histone acetyltransferases (HATs) known to date, CBP / p300 acetylates thousands of sites worldwide, many of which are hallmark histone sites in actively transcribed gene regions (Weinert et al., 2018). p300 / CBP are often referred to as a single entity because of their extensive homology and functional similarities (see Kouzarides 2007). p300 / CBP can also function as a transcriptional coactivator, i.e., a protein that connects transcriptional activators to components of the basal transcriptional apparatus (see Janknecht and Hunter, 1996). P300 can function as a transcriptional coactivator for a large number of signaling pathways, including the HIV-1 viral machinery and E1A recruitment by adenovirus.

[0152] The benefits of enhanced viral production can be obtained by increasing exogenous expression of global HATs such as p300 / CBP or attenuating HDACs, active competitors of p300. HDACs deacetylate the same sites as p300 / CBP (Li et al., 2014). Example 6. Generation of packaging cell lines with exogenous p300 expression.

[0153] Using a single pulse exponentially decaying Bio-Rad Gene Pulser, a vector containing the p300 coding region expressed under the control of the CMV promoter and the blasticidin resistance gene (Figure 6A) was transfected into confluent HEK 293F (fHEK) cells by electroporation. Selection medium containing blasticidin was added to the culture medium, and surviving cells containing stably integrated p300 were expanded.

[0154] FIG6A is a schematic diagram of the p300 integrated gene cassette. The p300 gene is flanked by a CMV promoter and a SV40 poly A tail. The blasticidin (Bsd) resistance gene cassette is located downstream of the p300 gene cassette, which contains a CMV promoter, a gene encoding Bsd, and a SV40 poly A tail. FIG6B shows the HDAC shRNA gene cassette. The HDAC shRNA is flanked by a U6 promoter for shRNA expression. The blasticidin (Bsd) resistance gene cassette is located downstream of the shRNA expression cassette containing a CMV promoter, a gene encoding Bsd, and a SV40 poly A tail. FIG6C shows the spliced ​​Xbp1 (Xbp1s) gene cassette. The Xbp1s gene is flanked by a CMV promoter and a SV40 poly A tail. The blasticidin (Bsd) resistance gene cassette is located downstream of the Xbp1s gene cassette, which contains a CMV promoter, a gene encoding Bsd, and a SV40 poly A tail. Example 7. Generation of packaging cell lines with knockdown of HDAC.

[0155] The U6-HDAC shRNA transfer vector (Fig. 6B) is combined with an auxiliary vector, a packaging vector and an envelope vector using polyethyleneimine for packaging of the lentivirus. The complex is added to HEK 293 cells. The supernatant is harvested after transfection and the lentivirus titer is determined by sandwich ELISA. fHEK is infected with lentiviral particles containing U6-HDAC shRNA. The transduced cells are grown in a selection medium containing Blasticidin and the surviving cells containing stably integrated U6-HDAC shRNA are amplified.

[0156] The endoplasmic reticulum (ER) can be further enhanced by activating the unfolded protein response (UPR) pathway. Under ER stress, unspliced ​​Xbp1 mRNA is cleaved by the activated stress sensor Ire1α to produce spliced ​​Xbp1 (Xbp1s). Translated Xbp1s is translocated to the nucleus and targets hundreds of genes encoding ER chaperones, foldases, and ER-associated protein degradation (ERAD). Enhanced Xbp1s can increase ER biogenesis in mammary epithelial cells (Sharmin et al., 2021). The benefits of enhancing ER function by increasing the expression of spliced ​​Xbp1 can be exploited to improve viral yields. Example 8. Generation of packaging cell lines with exogenously expressed spliced ​​Xbp1s.

[0157] A vector containing the Xbp1 splice coding region (Xbp1s) expressed under the control of the CMV promoter and the blasticidin resistance gene (see Figure 6C) was transfected into confluent HEK 293F (fHEK) cells by electroporation using a Bio-Rad Gene Pulser with a single pulse exponential decay. Selection medium containing blasticidin was added to the culture medium, and surviving cells contained stably integrated Xbp1s. Example 9: Protocol for selecting fused cells with high mitochondria and high ROS

[0158] To screen for different phenotypes of mitochondria and reactive oxygen species (ROS), Deep Red Reagent (a fluorescent probe used to measure cellular oxidative stress in cells) was used to stain the hybrids; TMRM (tetramethylrhodamine methyl ester), which is used to measure the membrane potential of mitochondria in living cells; and Biotracker 405Blue Mitochondria, which stains mitochondrial membranes. LIVE / DEAD Fixable NIR was used to stain living cells in this experiment.

[0159] Figure 8 The workflow used is shown. Confluent cells were cultured in complete, animal origin-free (AOF), chemically defined cell culture medium: CDM4 PerMAb + 6mM L-Glutamine and cultured using StemPro TM Accutase TM Cell Dissociation Reagent was used for separation. The cell sample was mixed with the calculated volume of each dye and the cell suspension (containing 1x108 cells) was adjusted to the final concentration in a 500 mL shake flask. The sample was incubated overnight in a shaker at 120 rpm under 8% CO2.

[0160] For cell sorting, centrifuge 200 mL of cell samples at 300 x g for 5 minutes. Suspend the cell pellet in Accutase cell dissociation reagent, dilute, and filter into a sterile 50 mL centrifuge tube. Use Sony SH800S Cell Sorter to sort cells, and the sorting gates are as follows: Gate 1-Cell ID Gate; Gate 2-Single Cell Gate; Gate 3-Live Cell Gate; Gate 4-Biotracker405Blue Mitochondria (select the top 10%); TMRE x CellROX Deep Red (select the top 10% quadrant).

[0161] 500,000 sorted cell populations were expanded for 4-5 days and then used for single-cell cloning in 96-well plates with 150 μl of medium per well. Once a single well reached 80% confluence, it was expanded stepwise to a 125 mL shake flask and used to create a cell bank. Example 10. Protocol for transient transfection of HEK 291 cells to express AAV capsids containing reporter genes

[0162] Useful sources of reagents are the AAV MAX system (available from ThermoFisher), AAV Transfection Kit and Takara's AAVpro helper-free system for various AAV serotypes.

[0163] One day before transfection, host cells were inoculated into 125-mL cell culture flasks at a density of 3x106 viable cells / mL in 25 mL of complete, animal origin-free (AOF), chemically defined cell culture medium (CDM4 PerMAb) containing 6 mM L-glutamine and grown to a density of 3.0x106 viable cells / mL with a viability of >90%. Cells were centrifuged at 300xg in a conical tube and resuspended in 25 mL of virus production medium. After transfer to a new shake flask, 250 μL of AAV-MAX Enhancer was added. Cells were cultured on an orbital shaker in a 37°C incubator until DNA / transfection complexation was complete.

[0164] Figure 8 Plasmids used to prepare AAV vectors containing an enhanced green fluorescent protein (EGFP) reporter gene for screening cell hybrids for virus production were mapped. A mixture of these plasmids was prepared as shown in Table 4.

[0165] Place 75 μL of Trans- Add to the plasmid in AAVCFS&E solution and incubate at room temperature for 15 minutes to form transfection complexes. Add 2.7 portions of this mixture to each 125 mL flask and return to the incubator for 12 to 18 hours. Add 0.3 mL of 0.5 M sodium butyrate and harvest the cells 72 hours after transfection. Incorporation by Reference

[0166] For all purposes in the United States, each publication and patent document mentioned in this disclosure is herein incorporated by reference in its entirety for all purposes to the same extent as if each individual publication or document was specifically and individually indicated to be incorporated by reference. Practice of the claimed invention

[0167] The technology and its use provided in this disclosure are described within the scope of the assumed understanding of the general principles of virus and drug manufacturing. These discussions are intended to inspire and interest the reader and are not intended to limit the practice of the claimed invention. All products and methods claimed in this application can be used for any suitable purpose without restriction unless otherwise specified or required.

[0168] Although the present disclosure has been described with reference to specific embodiments, changes may be made and equivalents may be substituted to adapt the present disclosure to a particular context or intended use, as a matter of routine experimentation, thereby achieving the benefits of the present disclosure without departing from the scope of what is claimed.

Claims

1. A method for producing a viral vector or particle, the method comprising: providing a starting population of cultured cells; forming cell hybrids from the starting population, each of the cell hybrids comprising two or more cells; genetically engineering the selected cell hybrid to express elements of the virus and the payload, The genetically engineered cells are cultured to produce viral vectors or particles containing the elements and encapsulating the payload.

2. A process for establishing a production cell line for efficiently producing viral vectors or particles, the process comprising: (a) providing a starting population of cultured cells; (b) forming cell hybrids from the starting population, each of the cell hybrids comprising two or more cells; (c) dividing the cell hybrid into a plurality of aliquots; (d) removing a cell sample from each of the aliquots; (e) genetically engineering cells in each of the samples to express elements of the virus and the reporter gene; (f) measuring the viral vectors or particles containing the reporter gene product produced by each of the samples, thereby determining which aliquots contain cell hybrids with higher functional titers of viral vectors or particles produced by cell hybrids than in other aliquots; (g) growing cells from one or more of said aliquots determined in step (f) to establish said producer cell line.

3. The process of claim 2, wherein the cell hybrid formed in step (b) is cloned in step (c), or wherein the cell measured in step (f) is cloned during or after step (g).

4. The process according to claim 2 or 3, wherein the reporter gene encodes a protein that emits a detectable signal, such as enhanced green fluorescent protein (EGFP).

5. The process according to claims 2 to 4, wherein step (c) comprises sorting or separating the cell hybrids according to the cell phenotype.

6. The process of claim 5, wherein the cellular phenotype comprises high or low intracellular levels of one or more organelles, such as mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus, or nucleolus, and / or one or more cytoplasmic or transcellular characteristics, such as reactive oxygen species (ROS), cellular redox, or pH.

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

8. A process according to claims 2 to 7, wherein step (f) comprises identifying which aliquots contain cell hybrids that produce more viral capsids per cell.

9. A process according to claims 2 to 8, wherein step (f) comprises identifying which aliquots produce a greater proportion of capsids filled with the reporter gene.

10. A method or process according to any preceding claim further comprising selecting a cell hybrid or cell aliquot having a higher growth rate than other cell hybrids.

11. A method or process according to any preceding claim, wherein the starting population is a single cell line.

12. A method or process according to any preceding claim, wherein the starting population is selected from: A cell line selected from the group consisting of CHO cells, mouse myeloma NSO cells, mouse myeloma SP2 / 0 cells, human embryonic kidney 293 (HEK 293) cells, baby hamster kidney 21 (BHK-21) cells, VERO cells, PER.C6 cells and HeLa cells; its autohybrids; and Hybrids of such cells with cells from another cell line or with primary cells.

13. A process for establishing a production cell line for efficiently producing viral vectors or particles, the process comprising: (a) providing a starting population of cultured cells; (b) forming cell hybrids from the starting population, each of the hybrids comprising two or more cells; (c) dividing the cell hybrid into a plurality of aliquots; (d) testing the aliquots or subpopulations thereof to determine which aliquots have higher mitochondrial and / or reactive oxygen species (ROS) content per cell; (e) growing cells from one or more of said aliquots determined in step (d) to establish said producer cell line.

14. A process according to claims 2 to 9 or claim 13, further comprising genetically engineering cells from the production cell line to express a transgene encoding one or more elements of a virus, thereby adapting the cells to produce viral vectors or particles containing the elements.

15. The process of claim 14, further comprising genetically engineering the cell to express a transgene encoding a payload, thereby adapting the cell to produce a viral vector or particle encapsulating the payload.

16. A production cell line suitable for efficient production of viral vectors or particles established according to the process of claims 2 to 9 or claims 13 to 15.

17. The producer cell line according to claim 16, which has been genetically modified to express a transgene encoding one or more elements of a virus, thereby adapting the cell to produce a viral vector or particle containing said elements.

18. A producer cell line wherein each cell is a hybrid of two or more parental cells from a starting cell line, wherein the producer cell comprises an expressible transgene encoding one or more elements of a virus, thereby configuring the cell to produce a viral vector or particle containing the elements.

19. The production cell line according to claim 17 or 18, wherein the cell further comprises a transgene constituting or encoding a payload, thereby adapting the cell to produce a viral vector or particle encapsulating the payload.

20. The production cell line of claim 19, wherein the payload comprises a nucleic acid configured to be expressed in vivo in a human subject upon administration to the subject.

21. The production cell line of claim 19, wherein the payload comprises a protein configured to be delivered into cells of the subject upon administration of the particle to the subject.

22. The production cell line of claim 19, wherein the payload comprises a nucleic acid or a protein configured for ex vivo transfection of cells.

23. The producer cell line of claims 18 to 22, wherein the producer cells are cultured to produce at least 500 or 5,000 viral vectors or particles per cell.

24. The production cell line of claims 18 to 23, which produces more viral vectors or particles, produces viral vectors or particles with a higher functional titer, or produces viral vectors or particles with a higher proportion containing the payload compared to cells from the starting cell population that have been genetically modified with the same transgene.

25. A method of manufacturing comprising culturing cells of the producer cell line according to any one of claims 19 to 24 to produce viral vectors or particles encapsulating the payload.

26. The method of claim 1 or 25, further comprising purifying the vector or particle from the cell culture and complexing the vector or particle to produce a pharmaceutical product such that the product is suitable for human administration.

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

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

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

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

31. A method, process or product according to any preceding claim, wherein the viral vector and / or viral particle is an adeno-associated viral (AAV) vector or particle.

32. Use of a method, process or product according to any one of claims 1 to 26 in the manufacture of a viral vector or particle for use in treating a human disease or condition.

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

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

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

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