HEK293 cell line suitable for serum-free suspension culture and its applications

Abstract

The present invention relates to a HEK293 cell line suitable for serum-free suspension culture and its applications. Specifically, the present invention relates to a method for producing a viral vector such as an adeno-associated viral vector using the cell line, and a method for screening such a cell line.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] All documents cited or referred to in this application, including but not limited to all literature, patents, and patent applications disclosed herein ("Application Cited Documents"), all documents cited or referred to in this application cited documents, and manufacturer's manuals, specifications, product specifications, and product pages of any products mentioned in this application or any Application Cited Documents, are all incorporated by reference into this application and may be used in practicing the invention. More specifically, all references are all incorporated by reference into this application as if each document were incorporated by reference. Any Genbank sequences mentioned herein are incorporated by reference into this application. This application claims priority to a Chinese patent application having application number 202111190063.4, filed on October 12, 2021, the entire contents of which are incorporated by reference herein. [Technical field]

[0002] Citation of any document in this application is not tantamount to an admission that such document is prior art to this application.

[0003] The present application relates to a method for screening HEK293 cell lines suitable for serum-free suspension culture, and to a HEK293 cell line screened using the method, in particular, a PowerS TM The present application relates to the screened cell lines, in particular PowerS TM The present invention further relates to a method of producing a viral vector, such as an adeno-associated viral vector, using -293. [Background technology]

[0004] Gene therapy and cell therapy are developing rapidly and have become the most promising direction for the development of biomedical science. The main purpose of gene therapy is to introduce foreign genes into target cells or tissues to replace, compensate, block or correct specific genes to achieve the goal of treating diseases. In gene therapy, 70%-80% of treatment regimens are achieved by viral vectors. Viral vectors are the key to realizing the delivery of therapeutic foreign genes to the target site, and they can be injected as drugs as they are or used to produce drugs.

[0005] For many serious and life-threatening diseases, gene therapy is currently the most effective and only treatment, and the indications for gene therapy are changing from rare / ultra-rare diseases to more common diseases. Therefore, larger-scale production of viral vectors for gene therapy is urgently needed in the industry.

[0006] Viral vectors for introducing foreign genes into target cells are mainly derived from viral vectors such as retroviruses, adenoviruses and adeno-associated viruses (AAVs), among which adeno-associated virus vectors are the most widely used. Currently, the mainstream AAV production uses HEK293 cells as production cells, and adherent cultures of HEK293 cells are cultured in systems such as square bottles and flat dishes with the addition of fetal bovine serum, and viruses are obtained by transient transfection and packaging of multiple plasmids. For cell and gene therapy development companies, the production method of transiently transfecting adherent cells HEK293 is suitable for preclinical research, IND declaration and clinical trial periods because of the limited demand for viral vectors. This production method requires the use of fetal bovine serum, which is expensive and has the risk of being contaminated by animal-derived viruses. When the cell yield needs to be increased, it mainly relies on increasing the number of cell culture units, which often causes problems such as large space occupied and heavy workload. When larger-scale AAV production is required, one solution is to culture the production cell line in suspension. Compared with adherent culture, suspension culture can not only increase cell density, but also culture cells in serum-free medium to reduce potential immunogenic contamination and animal-derived components in the final product, simplify downstream purification processes, and greatly promote the conversion of products to clinical levels. In addition, the scale-up of suspension culture can also reduce batch-to-batch differences compared to adherent culture due to the increase in culture units.

[0007] As described above, HEK293 cells are commonly used producer cells for AAV vectors. They are also called human embryonic kidney cells 293 and are a cell line derived from human embryonic kidney cells. They have characteristics such as high transfection efficiency and ease of culture. There are many problems in the suspension adaptation process of HEK293 cells, for example: (1) at present, the method of suspension adaptation mainly uses serum-reduced suspension adaptation, and the adaptation cycle is long, generally taking about 3 months; (2) in the suspension adaptation process, the phenomenon of HEK293 cells clumping is easy to occur, which makes many plasmids for vector production unable to transfect the cells, and the cells in the middle of the clump die due to lack of nutrition; at present, the main method to improve cell clumping is to add anti-clumping agents, but the addition of anti-clumping agents significantly reduces the transfection efficiency; (3) the culture medium has a significant effect on cell growth state, cell density and clumping, etc.; when HEK293 cells are adapted to different culture media, their growth expression is different; (4) in the process of subcloning, if the selected cell line is monoclonal, the phenomenon of unstable virus expression levels in cells of different passage numbers may occur when performing late toxin production test experiments.

[0008] At present, there are no precise regulations on the requirements for monoclonal origin in gene therapy, but monoclonal origin is one of the essential conditions for product quality and expression stability. Currently, the methods of screening monoclonal cells include: (1) performing two rounds of finite dilution and controlling the diluted cell density to less than 0.5 cells / well; (2) one round of finite dilution plus monoclonal imaging photography; (3) FACS plus one round of finite dilution or monoclonal imaging photography; and (4) semi-solid culture. The problem that exists is that the cell viability after FACS sorting is low, and HEK293 cannot be selected and photographed in semi-solid medium, and monoclonal origin data cannot be provided. The currently commonly used method is one round of finite dilution plus monoclonal imaging photography. Summary of the Invention

[0009] Through numerous experiments, the inventors of the present application have discovered a method for screening HEK293 cell lines suitable for serum-free suspension culture, which allows for screening of cell lines with high dispersibility and high growth density.

[0010] Thus, in one aspect, the present application provides a method for screening HEK293 cell lines suitable for serum-free suspension culture, the method comprising: i) taking HEK293 cells cultured in an adherent culture in a serum-containing medium and culturing them in suspension in a serum-free medium; and ii) selecting a monoclonal cell line suitable for serum-free suspension culture.

[0011] The serum-containing medium in step i) may contain 10% serum, for example 10% fetal bovine serum.

[0012] Step i) The medium suspension culture may comprise a shaking culture at 37° C. and 8% CO2.

[0013] The suspension culture in step i) may include passaging of the cells.

[0014] The serum-free medium may be selected from OPM-293 CD03 medium (Susumu Okuura, 81070-001), LV-MAX production medium (Gibco, A35834-01), and Expi293 expression medium (Gibco, A14351-01). In one embodiment, the serum-free medium is OPM-293 CD03 medium.

[0015] Step ii) may include a step of selecting monoclonal cells suitable for serum-free suspension culture by at least one round of finite dilution and photographic imaging, and a step of suspension culturing the selected monoclonal cells. In one embodiment, step ii) includes a step of selecting monoclonal cells suitable for serum-free suspension culture by two rounds of finite dilution and photographic imaging, and a step of suspension culturing the selected monoclonal cells.

[0016] Step ii) may further comprise testing the ability of the selected monoclonal cells to produce viral vectors, e.g. detecting the titer of the viral vector produced thereby.

[0017] The suspension culture of the selected cells in step ii) may include passaging of the cells.

[0018] In the process of carrying out the method, there is no need to add any anti-agglomerating agent.

[0019] In the method of the present application, 1) cells cultured in a serum-containing medium are directly cultured in a serum-free medium, and there is no step of gradually reducing serum, which greatly shortens the adaptation and screening process, and the adaptation and screening period can be shortened to 20 days; 2) cells are cultured using a serum-free medium, which avoids potential immunogenic substance contamination and animal-derived components, and simplifies the downstream purification process; 3) a serum-free medium selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium, especially OPM-293 CD03 medium, is used, which is more advantageous for screening HEK293 cell lines suitable for suspension culture, i.e., with high growth density and high dispersibility; 4) the use of anti-aggregation agents is avoided, and the transfection efficiency of cells is not affected; and 5) at least one round, for example two rounds, of finite dilution and imaging ensures the monoclonal origin, ensuring that the cells provide stable virus expression levels, while also providing intuitive data support for the monoclonal origin.

[0020] The present application also encompasses the use of a medium selected from LV-MAX production medium (Gibco, A35834-01), Expi293 expression medium (Gibco, A14351-01) and OPM-293 CD03 medium (Susumu Okuura, 81070-001) in screening for HEK293 cell lines with high dispersibility and high growth density. In one embodiment, the screening medium is OPM-293 CD03 medium.

[0021] Because the cell growth and aggregation states are not exactly the same in different media, the inventors of the present application found that HEK293 cells grew to a high density (~1 × 10 ) in OPM-293 CD03 medium, LV-MAX production medium, and Expi293 expression medium. 7 It was found that the OPM-293 CD03 medium had the best screening effect among the three media.

[0022] In another aspect, the present application relates to a HEK293 cell line screened by the above method.

[0023] The HEK293 cell line screened by the method of the present application can grow in suspension culture in serum-free medium. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium. In one embodiment, the serum-free medium is OPM-293 CD03 medium.

[0024] The cell line may not exhibit cell clumps during the suspension culture process. In one embodiment, the cell line does not aggregate in liquid medium.

[0025] The cell line may be used for the production of viral vectors, in particular adeno-associated viral vectors.

[0026] In particular, the present application relates to a novel human embryonic kidney HEK293 cell line PowerS, which was deposited with the China General Microorganism Collection Center (CGMCC) under the Budapest Treaty on August 9, 2021 and bears the deposit number CGMCC No.:23020. TM -293 provided. PowerS TM The -293 cell line can grow in suspension culture in serum-free medium, with high proliferation density, good viability, no cell clumps, i.e., high dispersibility, and therefore does not require the addition of additional anti-aggregating agents during the suspension culture process, and the cell line can produce high titer adeno-associated virus.

[0027] In yet another aspect, the present application relates to the use of the screened HEK293 cell lines in the production of viral vectors.

[0028] Specifically, the present application relates to the PowerS TM The present invention provides a method for producing a viral vector using a cell line comprising: i) culturing said cell line; ii) introducing a viral vector expression system into said cell line; iii) culturing said cell line under conditions in which the viral vector is produced; and iv) harvesting the viral vector.

[0029] Step i) may comprise suspension culture of the cell line. In one embodiment, the cell line is cultured in serum-free medium with shaking at 37° C. and 8% O2. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium. In one embodiment, the serum-free medium is OPM-293 CD03 medium.

[0030] Step i) may further comprise passaging the cell line.

[0031] Step ii) may comprise introducing the viral expression system into the cell line, for example by calcium phosphate co-precipitation, polyethylenimine (PEI)-mediated transfection. In one embodiment, the viral expression system is introduced into the cell line by polyethylenimine (PEI)-mediated transfection.

[0032] The viral vector may be an adeno-associated viral vector. The adeno-associated viral vector may be any serotype of adeno-associated viral vector, its chimera and hybrid. In one embodiment, the adeno-associated viral vector is selected from AAV2, AAV5, AAV8, AAV9 and AAV10.

[0033] The viral vector expression system may include a vector plasmid containing a heterologous nucleic acid, a packaging plasmid containing rep-cap, and a helper plasmid. The helper plasmid may include Ad5 genes including VA, E2A, and E4OEF6. The length of the heterologous nucleic acid in the vector plasmid is about 4.7 kb or less.

[0034] The heterologous nucleic acid can code for a target protein or a non-translated RNA. In one embodiment, the heterologous nucleic acid comprises an open reading frame that codes for a target protein or a non-translated RNA. The heterologous nucleic acid can code for a polypeptide or a protein and achieve overexpression of the polypeptide or protein. The heterologous nucleic acid can code for an shRNA and interfere with expression of a target gene. The heterologous nucleic acid can code for a gRNA and Cas9 and achieve knockout of a target gene. The heterologous nucleic acid can code for a gRNA and dCas9 and achieve endogenous overexpression of a target gene. The vector plasmid can include a promoter that guides expression of the heterologous gene, for example, a promoter that can achieve cell / tissue specific expression.

[0035] Step iii) may comprise culturing the cell line in suspension. In one embodiment, the cell line is cultured in suspension in serum-free medium. In one embodiment, the cell line is cultured in serum-free medium with shaking at 37° C. and 8% O2. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium. In one embodiment, the serum-free medium is OPM-293 CD03 medium.

[0036] Step iii) may involve passaging the cells.

[0037] In step iv), the viral vector may be collected by lysing the cells or the viral vector secreted by the cell line may be collected from the culture medium.

[0038] The method may further comprise subjecting the viral vector to titer detection, purification, etc. after step iv).

[0039] Other features and advantages of the present disclosure will be apparent based on the following specific descriptions and examples, which should not be construed as limiting. All references, GenBank accession numbers, patents and published patent applications cited in this application are hereby expressly incorporated by reference.

[0040] It should be noted that in this application, and particularly in the claims, terms such as "comprising," "including," and "consisting essentially of" allow for the presence of elements not expressly recited. Aspects and embodiments of the invention described herein include those aspects and embodiments that "comprise," "consist," and "consist essentially of."

[0041] As used in this specification and the appended claims, the singular forms "a," "one," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "one / one molecule" optionally includes a combination of two / two or more such molecules, and so forth.

[0042] As used in this application, the term "about" refers to a conventional margin of error for the relevant numerical value, which is readily known to one of ordinary skill in the art. Any numerical value or parameter referred to herein as "about" includes (describes) embodiments that relate to the numerical value or parameter itself. [Brief description of the drawings]

[0043] The following examples are given by way of example, but are not intended to limit the invention to the specific details of the embodiments described, and may be better understood in connection with the accompanying drawings, in which: [Figure 1] 13 is a growth curve of adherent HEK293 cells. [Diagram 2] FIG. 1 shows viral titers of adherent HEK293 cells packaging AAV2, AAV5, AAV8, and AAV9. [Diagram 3] Morphology of cells acclimatized in different serum-free media. [Figure 4] Growth curves of cells acclimatized in three types of serum-free media. [Diagram 5] Monoclonal derived screening diagram. [Figure 6] Morphology of monoclonal cells. [Figure 7] Cell growth / viability curves of clone 13, clone 20 and clone 29. [Figure 8] AAV2 viral titer yield plot of clone 13, clone 20 and clone 29. [Figure 9] AA5, AAV8 and AAV9 viral titer yield plots for Clone 13, Clone 20 and Clone 29. [Figure 10]FIG. 13 shows AAV5 viral titer yields of clone 13 at passages 5, 10, 15, and 20.

[0044] Description of deposit: The novel human embryonic kidney HEK293 cell line PowerS-293 in this application has been deposited at the Center for Ordinary Microorganisms, China Commission on Microbial Species Preservation and Management, under the Budapest Treaty, on August 9, 2021, with the address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, College, No. 1 Beichen West Road, Chaoyang District, Beijing, China.

[0045] The deposit number is CGMCC No.:23020. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Unless otherwise defined, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] The present application provides a method for screening a cell line suitable for serum-free suspension culture, and a cell line screened thereby, which can be used to produce highly dispersible, highly dense, and highly titered viral particle vectors, particularly adeno-associated viral vector particles.

[0048] Viral Vectors Viral vectors are tools used by molecular biologists to deliver genetic material into cells, which can carry foreign genes and be packaged into viral particles to mediate the transfer and expression of foreign genes without causing disease in living organisms. Known viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viruses, etc.

[0049] As used herein, the terms "viral vector," "viral particle," or "viral vector particle" are used interchangeably herein. The terms "heterologous nucleic acid," "heterologous gene," "foreign nucleic acid," or "foreign gene" refer to a nucleic acid or gene that is not naturally present in a virus. Generally, a heterologous nucleic acid or foreign gene comprises an open reading frame that encodes a target protein or a non-translated RNA.

[0050] Adeno-associated virus Adeno-associated virus (AAV), a member of the Parvoviridae family, is an autonomously replicating, non-enveloped, icosahedral parvovirus that contains a linear, single-stranded DNA genome of approximately 4.7 kb.

[0051] The AAV genome consists of two open reading frames, Rep and Cap, flanked by two 145 bp inverted terminal repeats (ITRs). The ITRs pair to form a hairpin structure, allowing primase-independent synthesis of a second complementary DNA strand. Rep and Cap can translate a variety of proteins, including Rep78, Rep68, Rep52, and Rep40, which are required for the AAV life cycle, and the capsid proteins VP1, VP2, and VP3.

[0052] As used herein, "adeno-associated virus" or "AAV" includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and any other AAV now known or later discovered. The genomic sequences of several AAV serotypes, as well as the sequences of the native ITRs, Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in common databases, such as GenBank.

[0053] The term "terminal repeat" or "TR" as used herein includes any viral terminal repeat or synthetic sequence that constitutes a hairpin structure and functions as an inverted terminal repeat (ITR), i.e. mediates necessary functions such as replication, viral packaging, integration and / or proviral rescue. The ITR may be an AAV ITR or a non-AAV ITR. Non-AAV ITR sequences, such as sequences from other parvoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that functions as the SV40 origin of replication, may be used as an ITR, which may be further modified by truncation, substitution, deletion, insertion and / or addition. Furthermore, the ITR may be partially or completely synthetic.

[0054] The AAV genome has palindromic sequences at its 5' and 3' ends. The palindromic nature of the sequence causes the formation of a hairpin structure, which is stabilized by the formation of hydrogen bonds between complementary base pairs. The hairpin structure may be considered to be in the shape of a "Y" or "T". The "AAV inverted terminal repeat" or "AAV ITR" may be derived from any AAV, including, but not limited to, serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and any other AAV now known or later discovered. AAV ITRs need not have their native terminal repeat sequences, e.g., the native AAV ITR sequences can be modified by insertions, deletions, truncations and / or missense mutations, provided that the terminal repeats mediate a required function, such as replication, viral packaging, integration and / or proviral rescue.

[0055] As one of the most commonly used viral vectors in gene therapy, AAV has the following characteristics:

[0056] 1. AAV is highly safe and has low immunogenicity. AAV is a replication-defective DNA virus that lacks the ability for autonomous replication, and wild-type AAV relies on the rep gene for low-frequency site-specific integration. Currently, there are no reports of human or mammalian diseases caused by AAV, and it is also one of the safest viral vectors in gene therapy drugs approved for sale by the FDA.

[0057] 2. AAV has a broad host range and is capable of infecting both dividing and non-dividing cells.

[0058] 3. AAV has strong diffusion ability. AAV has a diameter of about 20-26 nm, a small volume, high titer, and good diffusion ability. Some AAVs, such as AAV9, have the ability to pass through the blood-brain barrier and are widely applied in the field of neuroscience.

[0059] 4. AAV has the ability to maintain long-term gene transcription expression in vivo, with in vivo expression typically peaking at 3 weeks and continuing at high expression thereafter, with a duration of action exceeding 5 months.

[0060] 5. AAV has various serotypes. According to the cell receptors to which the capsid protein binds, AAV can be divided into various serotypes. In the body of primates, there are 13 types of AAV with different serotypes (i.e., AAV1-AAV13), of which AAV2, AAV3, and AAV9 are derived from humans. Based on these characteristics of AAV, different serotypes of AAV can be selected to transfect different target tissue types. In the case of the central nervous system, preferably AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9 are used; in the case of the heart, preferably AAV1, AAV8 and AAV9 are used; in the case of the kidney, preferably AAV2 is used; in the case of the liver, preferably AAV7, AAV8 and AAV9 are used; in the case of the lung, preferably AAV4, AAV5, AAV6 and AAV9 are used; in the case of the pancreas, preferably AAV8 is used; in the case of photosensitive cells, preferably AAV2, AAV5 and AAV8 are used; in the case of retinal pigment epithelial cells, preferably AAV1, AAV2, AAV4, AAV5 and AAV8 are used; and in the case of skeletal muscles, preferably AAV1, AAV6, AAV7, AAV8 and AAV9 are used.

[0061] Serotype 2, or AAV2, is the most widely studied and most widely applied AAV. At present, AAV2 has been found to bind to three cell receptors: heparan sulfate proteoglycan (HSPG), integrin aVβ5, and fibroblast growth factor receptor 1 (FGFR-1). HSPG is the main receptor, and the other two are co-receptors, which allow AAV2 to enter cells via receptor-mediated endocytosis. Research has shown that AAV2 can kill cancer cells without harming normal cells.

[0062] Other serotypes have a higher gene delivery efficiency than AAV2. As described above, AAV6 is better at infecting airway epithelial cells, AAV7 has a higher transfection efficiency for bone marrow muscle cells and is similar to AAV1 and AAV5, AAV8 is better at transfecting cardiomyocytes, AAV1 and AAV5 are highly efficient in gene delivery to vascular endothelial cells, most serotypes can transfect neural cells, and AAV5 can also transfect astrocytes.

[0063] Adeno-associated virus vector construction When constructing an AAV vector plasmid, the rep and cap genes can be removed and cloned into separate plasmids because the ITRs are the only cis-acting elements required for genome replication and packaging. A promoter and a heterologous gene driven by the promoter can be cloned between the two ITRs and efficiently packaged into the AAV capsid.

[0064] AAV lacks replication capacity and therefore requires co-infection with a helper virus, such as adenovirus (Ad) or herpes simplex virus (HSV). After co-infection with Ad or HSV, AAV utilizes some helper virus early genes to promote its self-replication. Infection of Ad into producer cells to produce AAV is effective for AAV production, but results in the production of excess Ad particles. Complete removal of Ad relies on physical techniques, such as CsCl gradients, column chromatography, and heat denaturation steps, to inactivate any remaining Ad particles that may still be present. Most of these procedures are successful to some extent, but the possibility of Ad infection is an unnecessary risk, and the presence of Ad denatured proteins is unacceptable for clinical use.

[0065] A significant improvement in the development of AAV production is the introduction of triple plasmid transfection. The method uses a variant of the rep and cap plasmids and the ITR plasmid, avoiding the use of Ad. Specifically, the Ad proteins E1A, E1B, E4 and E2A and the VA RNA are cloned into a single plasmid called XX680. By providing the Ad helper genes on the XX680 plasmid, the AAV vector is obtained from the transfected cells, avoiding the production of Ad. When transfected and produced using HEK293 cells, the helper plasmid is further simplified to contain only VA, E2A and E4OEF6, since HEK293 cells constitutively express E1A / 1B. The vector plasmid containing the ITR and heterologous nucleic acid, the packaging plasmid containing rep-cap, and the helper plasmid containing VA, E2A and E4OEF6 constitute the AAV vector expression system.

[0066] As used herein, "AAV expression system" refers to a system of one or more polynucleotides that, when introduced into a suitable host cell, is sufficient to support the production of AAV.

[0067] As used herein, the "Rep gene" or "Rep coding sequence" of AAV refers to a nucleic acid sequence that encodes AAV nonstructural proteins that mediate viral replication and the production of new viral particles. AAV replication genes and proteins are described, for example, in Fields et al., Virology, Vol. 2, Chapters 69 and 70 (4th ed., Lippincott-Raven Publishers). The "Rep coding sequence" does not need to encode all AAV Rep proteins. For example, in the case of AAV, the Rep coding sequence does not need to encode all four AAV Rep proteins (Rep78, Rep68, Rep52, and Rep40). The Rep coding sequence encodes at least those replication proteins necessary for viral genome replication and packaging into new viral particles. The Rep coding sequence generally encodes at least one large Rep protein (i.e., Rep78 / 68) and one small Rep protein (i.e., Rep52 / 40). In some embodiments, the Rep coding sequence encodes an AAV Rep78 protein and an AAV Rep52 and / or Rep40 protein. In other embodiments, the Rep coding sequence encodes a Rep68 and a Rep52 and / or a Rep40 protein. In still further embodiments, the Rep coding sequence encodes a Rep68 and a Rep52 protein, a Rep68 and a Rep40 protein, a Rep78 and a Rep52 protein, or a Rep78 and a Rep40 protein. As used herein, the term "large Rep protein" refers to Rep68 and / or Rep78. The large Rep protein may be wild-type or synthetic. The wild-type large Rep protein may be from any AAV, including, but not limited to, serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, and any other AAV now known or later discovered. The large synthesized Rep protein may be modified by insertions, deletions, truncations and / or missense mutations.

[0068] As used herein, an AAV "cap gene" or "cap coding sequence" encodes the structural proteins that form a functional AAV capsid (i.e., capable of packaging DNA and infecting a target cell). Typically, the cap coding sequence encodes all AAV capsid subunits, but may encode less than all capsid subunits, so long as a functional capsid is produced. Typically, the cap coding sequence is present on a single nucleic acid molecule. The capsid structure of AAV is described in detail by BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 and 70 (4th Edition, Lippincott-Raven Publishers).

[0069] A "vector plasmid" herein generally requires only the ITRs in cis to generate virus, with or without all other viral sequences, and may be provided in trans. Typically, AAV vector plasmids retain only one or more ITR sequences to maximize the size of the heterologous gene that can be effectively packaged by the vector. Structural and nonstructural protein coding sequences may be provided in trans (e.g., by a vector, e.g., a plasmid, or by stably integrating sequences into the packaging cell). In an embodiment of the present application, an AAV vector plasmid comprises at least one ITR sequence (e.g., an AAV ITR sequence), optionally two ITRs (e.g., two AAV ITRs), which are usually at the 5' and 3' ends of the vector genome and flank, but do not necessarily contiguous with, the heterologous nucleic acid. The ITRs may be the same as or different from each other.

[0070] As research progressed, it was discovered that AAVs of different serotypes could hybridize with each other, and the hybridized AAVs had both heterozygous characteristics, leading to the vigorous birth of AAV subtypes. Subtypes are usually produced by combining the genome of one serotype with the capsid protein of another serotype. Currently, the AAVs commonly used in research are hybrid virus vectors produced by combining the AAV2 genome with different capsid proteins, and are generally designated AAV2 / N (where N is a different capsid serotype). Recombinant viruses have the ability to stably express and incorporate AAV2, as well as acquire the tissue infection tropism of different serotypes, and exhibit a certain organ targeting specificity. For example, AAV2 / 1 has tissue affinity for the nervous system, muscle, skeletal muscle, cardiac muscle, and smooth muscle; AAV2 / 2 has tissue affinity for the retina, nervous system, muscle, liver, and vascular smooth muscle; AAV2 / 3 has tissue affinity for muscle, liver, lung, and eye; AAV2 / 4 has tissue affinity for the nervous system, muscle, eye, and brain; AAV2 / 5 has tissue affinity for the nervous system, lung, retina, liver, and synovial joint; AAV2 / 6 has tissue affinity for the nervous system, lung, muscle, and heart; AAV2 / 7 has tissue affinity for muscle and liver; rAAV2 / 8 has tissue affinity for the nervous system, liver, muscle, adipose tissue, pancreas, and retina; AAV2 / 9 has tissue affinity for the nervous system, cardiac muscle, lung, retina, and skin; AAV-PHP.eB can pass through the blood-brain barrier; and AAV-PHP.S can target all peripheral nerves. Research has shown that compared with AAV2, AAV2 / 5 can infect more brain cell types and has a higher infection accuracy. AAV-DJ has hybrid capsid proteins from eight different AAVs and can infect cells in many regions of the body. AAV can also remodel surface amino acid residues to further evade detection by the immune system.

[0071] 2. Construction of Viral Vectors The production of viral vector particles typically involves the following steps: 1) Cultivating and amplifying host cells, 2) introducing a viral vector expression system into the host cells, 3) culturing and amplifying the host cells containing the introduced viral vector expression system, and 4) collecting and purifying the produced viral vector.

[0072] Currently, some viral vectors (e.g., AAV vectors) can be packaged in vitro (cell-free), but such packaging systems still require cell extracts and have rather low packaging efficiency, which is not yet at a production level.Up to now, packaging of viral vectors has mainly been carried out in host cells that are sensitive to the virus.Host cells not only provide the environmental conditions for viral replication and packaging, but also many cellular components are directly involved in the process of viral replication and packaging.

[0073] There are two types of host cells for virus production: one is a cell line that stably expresses viral packaging system elements, and the other is a transiently transfected cell line. The transient transfection packaging method is easy to operate and time-saving, and is the most commonly used method for producing viral vectors.

[0074] Host cells that stably express viral packaging elements are also referred to as producer / packaging cell lines. In the context of adeno-associated vectors, the term "packaging cell line" may refer to a cell line into which the rep and cap genes have been introduced, and "producer cell line" may refer to a cell line into which the rep and cap genes, ITRs, and a foreign gene have been introduced. Packaging cell lines require co-transfection of AAV vector plasmids and helper plasmids to produce AAV, whereas producer cell lines can produce AAV by transfection of only the helper plasmid. In one embodiment, a "packaging cell line" is a cell line into which the rep and cap genes have been inserted into the cell genome, and a "producer cell line" is a cell line into which the rep and cap genes, ITRs, and a foreign gene have been inserted into the cell genome. In another embodiment, a "packaging cell line" is a cell line into which the rep and cap genes have been stably inserted into the cell genome, and a "producer cell line" is a cell line into which the rep and cap genes, ITRs, and a foreign gene have been stably inserted into the cell genome. Stably transfected cell lines for AAV are usually HeLa cells.

[0075] Transient transfection production of AVV is commonly performed using HEK293 cells, also called human embryonic kidney 293 cells. These cells constitutively express E1a / b, one of the functional adenoviral genes required for AAV replication.

[0076] The first step in the production of viral vector particles is to culture and amplify host cells. Host cell culture is generally divided into adhesion culture and suspension culture. To increase the production of viral vectors, the total surface area that cells can attach to can be increased, or suspension culture of cells can be performed.

[0077] As the most commonly used host cell for AAV production, HEK293 cells are usually grown adherently in cell culture flasks containing 10% fetal bovine serum, resulting in low viral yields. To increase the surface area on which cells can attach, we used HYPERFlask TM Multi-layer vessels such as the HYPERFlask (Corning) and multi-layer cell factory systems such as the cell factory system Numc EasyFill (ThermoScientific, Waltham, MA, USA) may be used. TM (Corning) has a total growth area of ​​approximately 1720 cm 2 It has a breathable surface, allowing for the exchange of oxygen and carbon dioxide, and has a yield approximately 10 times higher than that of a T-150 culture flask. The number of layers in a cell factory system (Numc, EasyFill, ThermoScientific, Waltham, MA, USA) can be as high as 40, creating a culture surface area equivalent to approximately 170 T-150 culture flasks. iCELLis (Pall Life Sciences, Hoegaarden, Belgium) and Scale-X TM Fixed-bed bioreactors such as the Bioreactor System (Univercells, Gosselies, Belgium) have also been developed, where polyethylene terephthalate (PET) fibers or a layer of PET fibers is packed onto a fixed bed to provide a large surface area for cell attachment. The cell growth area with the iCELLis500 is approximately 3000 HYPERFlasks.

[0078] Various equipment updates have made it possible to expand serum-dependent adherent HEK293 cells, but this has taken time and effort. Therefore, researchers have begun to attempt to adapt HEK293 cells to be suitable for suspension culture. HEK293E cells were first adapted and cultured in suspension in serum-free medium. Then, new cell lines such as HEK293SF-3F6 were developed one after another (Ansorge, S. et al., (2009) Development of a Scalable Process for High-Yield Lentiviral Vector Production by Transient Transfection of HEK293 Suspension Cultures. J. Gene Med. 11: 868-876). Compared to adherent culture, suspension culture has various advantages, such as not requiring complicated instruments, not requiring mechanical force or enzymes to remove the cell wall, being easier to subculture, and being easier to scale up.

[0079] For cell and gene therapy development companies, the demand for viral vectors during preclinical research, IND filing and clinical trials is limited, so most of them produce adeno-associated virus vectors by the method of transient transfection of adherent cells HEK293. The adherent culture of HEK293 cells is usually carried out in the form of a cell factory, and the production method mainly increases the cell amount by increasing the number of culture units. Therefore, if more viral vectors are required, more space needs to be occupied and the production labor is also great. One of the ways to obtain a large growth area and reduce the amount of work is to adapt the cell line producing the adeno-associated virus to suspension culture. Compared with adherent culture, suspension culture can greatly improve the cell density.

[0080] The second step in producing viral vector particles is to introduce the viral vector expression system into a host cell, i.e., transfect the host cell with a packaging nucleic acid vector, a helper nucleic acid vector, a nucleic acid vector carrying a heterologous gene, etc.

[0081] "Viral expression system" herein refers to a system of one or more polynucleotides, which when introduced into a suitable host cell, is sufficient to support viral replication and packaging. The viral expression system may further include a heterologous gene, and the nucleic acid encoded thereby, e.g., RNA or polypeptide / protein, can be packaged together into a viral vector particle. An AAV expression system typically includes polynucleotides encoding AAV rep and cap, helper genes, and rAAV genome (e.g., ITR). "Nucleic acid vector" herein refers to a DNA or RNA vector, and may be a plasmid, or a bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), P1-derived artificial chromosome (PAC), fosmid, or cosmid, etc., in the embodiments for carrying out the invention. In the nucleic acid vector, an origin of replication, a promoter, a transcriptional regulatory element, etc. may be included as necessary. Any element may be operably linked to a promoter so that expression can be controlled. The promoters referred to herein may comprise known promoters (complete or partial), which may be stably activated or inducible, for example, in the presence of regulatory proteins. In one embodiment, the nucleic acid vector also comprises an efficient promoter, for example, a CMV promoter. Such promoters have the advantage of promoting high-level expression of elements encoded on the non-mammalian nucleic acid vector. In another embodiment, the CMV promoter comprises a sequence derived from human cytomegalovirus strain AD169. The sequence can be obtained from Genome Accession No. X17403, for example, base pairs 173731 to 174404. In the present application, a nucleic acid vector carrying a foreign gene may be referred to as a vector plasmid. A vector plasmid usually contains a promoter (e.g., CMV), a 3'ITR, a 5'ITR, and a potential foreign gene linked to the promoter.

[0082] "Transfection" or "infection" as used herein refers to the introduction of foreign genetic material, such as the "viral expression system" as described herein, into a host cell. Commonly used transfection methods are known to those skilled in the art, including but not limited to physical methods (e.g., electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, ballistic transfection, magnetofection, gene gun or particle bombardment), chemical reagents (e.g., calcium phosphate, highly branched organic compounds or cationic polymers) or cationic lipids (e.g., lipofection). Many transfection methods require contacting a plasmid DNA solution with cells and growing them to select for marker gene expression.

[0083] The introduction of foreign nucleic acid or nucleic acid vector into host cells is not easy, and must cross the barrier of the cell membrane, and high transfection efficiency is required to produce high titer viral vector particles. Methods of viral plasmid transfection are broadly divided into two categories: chemical and physical. Common chemical methods include calcium phosphate co-precipitation, polyethyleneimine (PEI)-mediated transfection, and Lipofectamine (Invitrogen)-mediated transfection, and physical methods include electroporation, such as flow electroporation, microinjection, and gene gun.

[0084] Calcium phosphate co-precipitation is suitable for various cell types, allows large numbers of cells to be transfected simultaneously, and can be used for large-scale virus production. However, calcium phosphate is sensitive to changes in pH value and highly toxic to cells. To avoid toxicity, it is necessary to culture the host cells in a medium containing serum or albumin, and to change the medium in a timely manner after transfection.

[0085] PEI-mediated transfection is as efficient as calcium phosphate. When using this method, it is necessary to select an appropriate PEI / DNA ratio. PEI is also toxic to cells, but it is not necessary to change the medium after transfection. In addition, the PEI method is not sensitive to pH value. The PEI method can be used for cells that are cultured in adhesion and suspension, and serum in the medium is essential (Toledo, JR et al., (2009) Polyethylenimine-Based Transfection Method as a Simple and Effective Way to Produce Recombinant Lentiviral Vectors. Appl. Biochem. Biotechnol.157:538-544).

[0086] Lipofectamine-mediated transfection is the most efficient and convenient, suitable for a variety of cell types, and has a very high transfection rate, e.g., in some cases, 100% of cells are seen to be transfected by adeno-associated virus. However, for large-scale viral vector production, the cost is high when using Lipofectamine.

[0087] Flow electroporation is as efficient as the calcium phosphate method, but requires less than one-third the amount of DNA, is non-toxic to cells, and is suitable for cells grown in suspension.

[0088] It has been reported that the addition of sodium butanoate to the medium after transfection can increase the virus titer.

[0089] Among the above transfection methods, calcium sulfate co-precipitation is more suitable for cells cultured in adhesion, such as HEK293 cells, flow electroporation is more suitable for cells cultured in suspension, and Lipofectamine is more suitable for small-scale applications due to cost reasons. In this application, PEI is selected to mediate the transfection of HEK293 cells with nucleic acid vectors.

[0090] Upon entering the host cell, some nucleic acid vectors randomly integrate into the endogenous genome of the mammalian host cell. Therefore, it is necessary to select host cells in which the nucleic acid encoded on the nucleic acid vector has been integrated, for example, using an antibiotic resistance selection marker, such as a zeocin resistance marker. Those skilled in the art will know how to promote integration of a nucleic acid vector, for example, how to linearize the nucleic acid vector, for example, a plasmid, if it is naturally circular. The nucleic acid vector may further include a region that shares homology with an endogenous chromosome of the host cell to guide the selected site integrated into the endogenous genome. It should be noted that the recombination site, if present on the nucleic acid vector, can be used for targeted recombination. Other targeted integration methods are well known in the art. For example, methods that induce targeted breaks in genomic DNA can be used to promote targeted recombination at a selected chromosomal locus. These methods usually involve inducing double-strand breaks (DSBs) or incisions in the endogenous genome using engineered breakage systems to repair the breaks by natural processes (e.g., non-homologous end joining) or repair templates (i.e., homology-directed repair or HDR). Cleavage can be achieved using specific nucleases such as engineered zinc finger nucleases (ZFNs), transcription activators (e.g., effector nucleases) (TALENs), using the CRISPR / Cas9 system and engineered crRNA / tracr RNA ('single guide RNA') to guide specific cleavage, and / or using nucleases based on the Argonaute system (e.g., derived from T. thermophilus and called 'TtAgo', Swarts DC et al., (2014) DNA-guided DNA interference by aprokaryotic Argonaute. Nature. 507(7491):258-261). Targeted cleavage using one of these nuclease systems can utilize HDR or NHEJ mediated processes to insert the nucleic acid into a specific target location.Thus, in one embodiment, at least one nuclease is used to integrate a nucleic acid sequence encoded on a nucleic acid vector into the genome (i.e., an endogenous chromosome) of a host cell, where the at least one nuclease cleaves the genome of the host cell and integrates the nucleic acid sequence into the genome of the cell. In another embodiment, the at least one nuclease is selected from a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), a CRISPR / Cas nuclease system, and combinations thereof.

[0091] Transfection can also be divided into transient transfection and stable transfection. In the case of transient transfection, the foreign gene is expressed but is not integrated into the cell genome and is not replicated. The expression time of the foreign gene that is transiently expressed in the cell is limited, and usually lasts only a few days or months before the foreign gene is lost due to various factors in the cell division process. Stable transfection is based on transient transfection and requires only one important accidental process, namely, in a small number of transfected cells, the foreign gene can be integrated into the cell genome. The foreign gene becomes part of the cell genome and is replicated, which is a marker of a stably transfected cell. The descendant cells of the stably transfected cell also express the foreign gene, thereby forming a stable cell line.

[0092] Transient transfection generally lasts for several days, and transient transfection is used to study gene expression, and cells are usually harvested within 24-96 hours after transfection, the specific time depending on various factors such as cell type, vector construction, etc. For this reason, transient transfection is generally used for short-term expression of genes or gene products, gene knockout or RNA-mediated gene silencing studies, and small-scale synthesis of proteins. Transient transfection of mRNA produces results faster than traditional transfection of plasmid DNA, because mRNA can be directly expressed outside the nucleus, and in some systems, mRNA can be expressed within minutes after transfection. In contrast, stable transfection must be performed when long-term gene expression is required, such as large-scale protein synthesis, long-term pharmacological studies, gene therapy studies, and long-term studies of genetic regulatory mechanisms. Stable transfection is longer and more laborious than transient transfection.

[0093] Stably transfected cell lines of AAV are usually Hela cells, e.g., the Hela S3 cell line, in which the rep and cap genes, or the rep and cap coding genes, may be introduced, or AAV can be produced by co-transfection of an AAV vector plasmid and a helper plasmid, in which the rep and cap genes, ITRs and a foreign gene may be introduced, or AAV can be produced by transfection of the helper plasmid alone.

[0094] Due to the difficulty of obtaining stably transfected cell lines, current mainstream virus production, for example adeno-associated virus vector production, still uses transient transfection of suspension cultured HEK293 cell lines.

[0095] The third step in viral vector production is similar to the first step, in which host cells transfected with a viral vector expression system are cultured and amplified under conditions that produce viral vector particles.

[0096] The fourth step is to isolate and purify the viral vector.

[0097] In one embodiment, the viral vector may be collected by lysing the cells, e.g., by removing the cells from the medium and then sedimenting the cells. In another embodiment, the viral vector may be collected from the cell medium, e.g., by isolating the vector secreted by the cells. To collect the AAV, some or all of the medium from the culture may be collected one or more times, e.g., periodically (e.g., every 12, 18, 24, or 36 hours) during the culture step, or, e.g., starting to collect the medium about 48 hours after transfection. After the medium is collected, fresh medium, with or without additional nutritional supplements, may be added to the culture. In one embodiment, the cells may be cultured in a perfusion system such that the medium flows continuously over the cells and is collected to isolate the secreted AAV. The AAV may be collected from the medium continuously, e.g., 48, 72, 96, or 120 hours or more after transfection, as long as the transfected cells maintain viability. In some embodiments, some secreted AAV serotypes, such as AAV8 and AAV9, are collected from the medium, and these AAV serotypes are not bound to host cells or are loosely bound to host cells.

[0098] The collected cell culture medium containing the viral vector particles may contain various impurities, such as host cells, other proteins produced by the host cells, plasmid DNA, serum, etc., where serum is complex in composition and may contain endotoxins and immunogenic proteins, etc. These impurities must be removed and the final product must be concentrated. In these processes, the biggest problem is how to maintain the activity / functionality of the viral vector, and therefore the process must be completed in the shortest possible time with as few steps as possible. It has become clear that the methods used for small-scale purification are not suitable for large-scale production. Some purification steps, such as centrifugation, do not meet cGMP standards, and high-speed centrifugation destroys the viral envelope to some extent.

[0099] Conventional large-scale viral vector purification methods include clarification, concentration and purification.

[0100] Clarification refers to the removal of host cells and host cell debris after collecting the supernatant. Small-scale clarification can be performed by methods such as centrifugation and microfiltration, while large-scale clarification is preferably performed using a 45 μm porous membrane. To avoid clogging of the wells, membranes with gradually decreasing pore sizes can be used to improve filtration efficiency.

[0101] The clarified viral vector particles can be concentrated and permeated with a suitable buffer using tangential flow filtration (TFF), which involves using a membrane with a pore size of 1 to 100 nm. This technology can significantly reduce the volume in a short time to concentrate the viral particles, remove serum, and degrade DNA fragments, etc. Most importantly, the operation by TFF is fully compliant with cGMP standards. Because the liquid in the TFF operation is parallel to the filter, the membrane clogging problem, which has been a problem in other ultrafiltration methods, can be significantly reduced, and the liquid flow rate can be maintained at a high level. Approximately 90 to 100 adeno-associated viral particles can be recovered by TFF. Currently, TFF can process about 100 L of viral product (Valkama, AJ et al., (2020) Development of Large-Scale Downstream Processing for Lentiviral Vectors. Mol. Ther. Methods Clin. Dev. 17: 717-730).

[0102] For pharmaceutical products applied to humans, the viral vector product needs to be further purified by chromatography. Anion exchange chromatography (AEX) is an efficient tool for viral vector purification based on the characteristic that viral vectors are positively charged at neutral pH. When the viral vector supernatant passes through a column of negatively charged matrix, the positively charged viral particles bind to the negatively charged matrix, and the impurities flow directly through the column. Then, the viral vector is exposed to a high salt environment (0.5-1M NaCl) to elute the bound particles from the anion exchange column. High salt may inactivate the viral particles, which is the only drawback of the technique. The viral particles can be exposed to a high salt elution solution at room temperature, and 50% of the virus can be inactivated (Segura, MDLM et al., (2005) A Novel Purification Strategy for Retrovirus Gene Therapy Vectors Using Heparin Afinity Chromatography. Biotechnol. Bioeng. 90: 391-404).

[0103] Affinity chromatography is a purification technique used to isolate biomolecules based on the specific interaction between the target molecule and a ligand attached to a chromatographic column. Due to its high molecular selectivity, the technique can simplify the purification steps to a certain extent. Affinity chromatography can be divided into several classes according to the difference in the interaction, such as affinity chromatography based on hydrogen bonds, electrostatic action, van der Waals forces, or antibody-ligand. The interaction between the antibody and the ligand is the most selective, and no viral vector purification by affinity chromatography using an antibody that binds to the envelope protein has been observed. Heparin is an affinity ligand that is cheap and interacts with various virus types, and has been used, for example, in the purification of adeno-associated viral vectors, and can recover up to 53% of the viral vector and remove up to 94% of protein impurities and 56% of residual DNA. Between the vector and heparin, there is an interaction between the positively charged molecules on the surface of the viral particle and the negatively charged heparin, and therefore NaCl is usually used to elute the vector from the heparin column. Typically, to elute the viral vector, the salt concentration needs to reach about 0.5 M, which requires the addition of additional washing or purification steps, such as the addition of additional TFF, to remove the salt.

[0104] In large-scale viral vector purification, size exclusion chromatography (SEC) can act as a cleaning step and effectively remove impurities that are left behind. SEC, also known as gel filtration chromatography, is based on the difference between the large volume of viral particles and the small volume of impurities when used in viral vector purification. All impurities are smaller than the pore size and therefore can pass through the column, leaving only the viral particles with a large volume. The disadvantage of SEC is that it is difficult to scale up and the loading volume is only 10% of the column volume. In addition, SEC purification requires a linear low flow rate and has a long operation time.

[0105] Regarding the purification of AVV, cesium chloride density gradient centrifugation was first used. Such a method is time-consuming and labor-intensive, has a low recovery rate, and cesium chloride has potential toxicity to the human body. Gimm et al. developed a purification method of immunoaffinity chromatography using specific antibodies against AAV structural proteins in 1998, which allows the cell lysate to be further purified, and about 70% of AAV can be recovered, with a purity of about 80%. AAV can be purified by heparin affinity chromatography, since heparin is an analogue of the natural receptor of AAV. A potential problem with such a method is that many proteins also bind to heparin. One method is to first perform density gradient centrifugation with iobitridol, and then perform heparin affinity chromatography, which allows about 50-70% of AAV to be recovered quickly and easily, with a purity of about 99%. Another method is to first lyse the cells with deoxycholate, then centrifuge to obtain the supernatant, and then heat at 56 °C for 45 minutes, denaturing the proteins, then centrifuging to remove the precipitate, and then performing heparin affinity chromatography. The method is also rapid and highly reproducible, with a recovery rate of more than 70% and a purity of up to 99%. In addition, a laboratory has proposed a three-step purification method using chloroform treatment-PEG / NaCl precipitation-chloroform extraction, taking advantage of the chloroform-resistant properties of AAV.

[0106] After the purification step, the viral vectors should be stored frozen at -80°C.

[0107] In this application, specifically, PowerS TM The present invention provides a method for producing viral vector particles using a cell line of the present application, including -293, the method comprising: i) culturing said cell line; ii) introducing a viral vector expression system into said cell line; iii) culturing said cell line under conditions in which viral vector particles are produced; and iv) harvesting the viral vector particles.

[0108] Step i) may comprise suspension culture of the cell line. In one embodiment, the cell line is cultured in serum-free medium with shaking at 37° C. and 8% O2. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium. In one embodiment, the serum-free medium is OPM-293 CD03 medium.

[0109] Step i) may further comprise passaging the cell line.

[0110] Step ii) may include introducing the viral expression system into the cell line by physical or chemical transfection methods. Physical transfection methods may include electroporation, microinjection, gene gun, etc. Chemical transfection methods may include calcium phosphate co-precipitation, polyethyleneimine (PEI)-mediated transfection, and Lipofectamine (Invitrogen)-mediated transfection, etc.

[0111] The viral vector may be an adeno-associated viral vector. The adeno-associated viral vector may be any serotype of AAV, chimera and hybrid, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and any chimera and / or hybrid thereof, such as AAV1-13. In one embodiment, the adeno-associated viral vector is, for example, AAV2, AAV5, AAV8, AAV9 and AAV10.

[0112] The viral vector expression system may include a vector plasmid containing a heterologous nucleic acid, a packaging plasmid containing rep-cap, and a helper plasmid. The helper plasmid may contain Ad5 genes including VA, E2A, and E4OEF6. The length of the heterologous nucleic acid in the vector plasmid is 4.7 kb or less.

[0113] In one embodiment, a method is used to generate an adeno-associated viral vector, comprising introducing into a cell line three nucleic acid vectors, each of which contains a rep-cap gene, an Ad5 gene (e.g., VA, E2A, and E4OEF6), and a heterologous gene between two ITRs.

[0114] The heterologous nucleic acid can code for a target protein or a non-translated RNA. In one embodiment, the heterologous nucleic acid comprises an open reading frame that codes for a target protein or a non-translated RNA. The heterologous nucleic acid can code for a polypeptide or a protein and achieve overexpression of the polypeptide or protein. The heterologous nucleic acid can code for an shRNA and interfere with expression of a target gene. The heterologous nucleic acid can code for a gRNA and Cas9 and achieve knockout of a target gene. The heterologous nucleic acid can code for a gRNA and dCas9 and achieve endogenous overexpression of a target gene. The vector plasmid can include a promoter that guides expression of the heterologous gene, for example, a promoter that can achieve cell / tissue specific expression.

[0115] Step iii) may include suspension culture of the cell line. The cell line may be cultured under any conditions suitable for the cell line to produce the viral vector, which may be determined by one skilled in the art. In one embodiment, the cell line is cultured in serum-free medium with shaking at 37°C and 8% O2. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium, and OPM-293 CD03 medium.

[0116] Step iii) may further comprise passaging the cells.

[0117] This application relates to a PowerS TM-293 host cells are used in bench and pilot batch production to produce viral vectors, for example in 300 ml shake flasks and in suspension culture in 1 L, 2 L, 10 L and 50 L bioreactors (e.g., WAVE reactors or glass reactors).

[0118] In step iv), the viral vector may be collected by lysing the cells or the viral vector secreted by the cell line may be collected from the culture medium.

[0119] The method may further comprise subjecting the viral vector to titer detection, purification, etc. after step iv).

[0120] Viral vector applications The viral vector expression system may include a nucleic acid vector containing a heterologous nucleic acid, for encoding a therapeutic or research protein, or a therapeutic or research non-translatable RNA, such as siRNA. The application of the viral vector depends mainly on the protein and nucleic acid that the heterologous nucleic acid encodes.

[0121] The heterologous nucleic acid can encode a therapeutic molecule, such as a therapeutic peptide, or a therapeutic nucleic acid, such as a siRNA. A "therapeutic molecule" can be a peptide or protein that can alleviate or relieve symptoms due to a protein deficiency or defect in a cell or subject. Alternatively, a "therapeutic" peptide or protein encoded by a heterologous nucleic acid is a substance that provides a benefit to a subject, for example, correcting a genetic defect, correcting a gene (expression or function) defect, or providing an anti-cancer effect. Therapeutic nucleic acids can include, for example, siRNAs, antisense molecules, and miRNAs. A heterologous nucleic acid can encode multiple useful products.

[0122] The heterologous nucleic acid can encode a hormone, growth factor, or differentiation factor, such as insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), platelet-derived differentiation factor (DRF), and the like. These include, but are not limited to, any member of the transforming growth factor beta superfamily, including transforming growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), TGF beta, agonists, inhibitors, or any member of the bone morphogenetic proteins (BMPs) BMP1-15, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factors NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), aggregation proteins, spindle protein-1 and spindle protein-2, hepatocyte growth factor (HGF), erythrophysic acid, noggin, and tyrosine hydroxylase.

[0123] Other useful heterologous nucleic acid products include proteins that regulate the immune system, including, but are not limited to, cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-17, monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β and γ, stem cell factor, flk-2 / fFlt3 ligand, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, G protein-coupled receptors (GPCRs), such as CCR5, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. Useful heterologous nucleic acid products further include regulatory proteins, such as complement regulatory proteins, membrane accessory proteins (MCPs), decay accelerating factors (DAFs), CR1, CF2 and CD59.

[0124] Other useful heterologous nucleic acid products include gene products capable of correcting inborn errors of metabolism, such as carbamoyl synthetase I, ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, clotting factors, such as factor V, factor VIIa, factor VIII, factor IX, factor X, factor XIII, or factor VIIIa. Examples of such cDNA include, but are not limited to, protein C, cystathionine-β-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl A dehydrogenase, propionyl A carboxylase, propionyl A carboxylase, glutaryl A dehydrogenase, insulin, β-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, glycine decarboxylase (CFTR) sequence, and dystrophin cDNA sequence.

[0125] Other useful heterologous nucleic acid products include those that can provide a defective, deficient or absent function or activity, such as, for example, antibodies, retinal pigment epithelium specific 65 kDa protein (RPE65), erythropoietin, low density lipoprotein receptor, lipoprotein lipase, ornithine carbamoyltransferase, β-globulin, α-globulin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfatases, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase (HGT), and the like. silyltransferase, β-25 glucocerebrosidase, sphingophospholipase hexosaminidase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors (e.g., insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factors 3 and 4, brain-derived neurotrophic factor, glial cell line-derived growth factor, transforming growth factor α and β, etc.), cytokines (e.g., α interferon, β interferon, interferon γ, interleukin 2, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, interleukin 14, interleukin 15, interleukin 16, interleukin 17, interleukin 18, interleukin 19, interleukin 20, interleukin 21, interleukin 22, interleukin 23, interleukin 24, interleukin 25, interleukin 26, interleukin 27, interleukin 28, interleukin 29, interleukin 30, interleukin 31, interleukin 32, interleukin 33, interleukin 34, interleukin 35, interleukin 36, interleukin 37, interleukin 38, interleukin 39 ... cytosine 12, granulocyte macrophage colony stimulating factor, lymphotoxin, etc.), suicide gene products (e.g., herpes simplex virus thymidine kinase, cytosine deamidase, diphtheria toxin, cytochrome P450, deoxycytidine cytokinase, tumor necrosis factor, etc.), drug resistance proteins (e.g., for providing drug resistance in cancer treatment), tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis (APC)), immune modulating peptides, resistance or immunogenic peptides or are proteins Tregitopes, or hCDR1, insulin, glucokinase, guanylate cyclase (LCA-GUCY2D), Rab satellite protein 1, LCA5, ornithine ketoacid transaminase, retinoschisis 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: linked retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2,A gene defect due to polycystic kidney disease (e.g., sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, saposin, etc.), one or more zinc finger nucleases for genome editing, or a donor sequence as a repair template for genome editing.

[0126] Alternatively, the heterologous nucleic acid may include, for example, siRNA, antisense molecules, and miRNA. Clinically useful adeno-associated virus vectors can express antisense genes for human immunodeficiency virus (HIV) (Levine BL et al., (2006) Gene transfer in humans using aconditionally replicating lentiviral vector. Proc Natl Acad Sci US A.103(46):17372-7) and other important human pathogens. These and other useful applications of adeno-associated virus and related vectors are discussed and incorporated by reference in Naldini (Naldini L. (2011) Ex vivo gene transfer and correction for cell-based therapies. Nat. Rev. Genet.12:301-315). Antisense genes contained in adeno-associated virus vectors can suppress expression of the following: the huntingtin (HTT) gene, genes associated with dentatorubral-pallidoluysian atrophy (e.g., atrophin 1, ATN1), X-chromosome androgen receptor in spinal-bulbar muscular atrophy, human Ataxin-1, -2, -3 and -7, Cav2.1P / Q voltage-gated calcium channel encoded by (CACNA1A), TATA-binding protein, Ataxin 8 inverse chain also called ATXN8OS, serine / threonine protein phosphatase 2A 55 kDa regulatory subunit Bβ isoform in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17), FMR1 in fragile X syndrome (Fragile X syndrome) mental retardation 1), FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile X mental retardation, myotonic protein kinase (MT-PK) in myotonic dystrophy, motor protein in Friedreich's ataxia, mutations in the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis, genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease,Apolipoprotein B (APOB) and Bacillus subtilis protease precursor protein convertase / kexin type 9 (PCSK9), excess blood cholesterol, HIVTat in HIV infection, human immunodeficiency virus transactivator of transcription gene, HIVTAR in HIV infection, human immunodeficiency virus transactivator response element gene, CC chemokine receptor (CCR5) in HIV infection, Rous sarcoma virus (RSV) in RSV infection, liver-specific microRNA (miR-) in hepatitis C virus infection, 122), p53, acute kidney injury, or delayed graft function following kidney transplantation, or kidney injury acute kidney failure, protein kinase N3 (PKN3) in advanced or metastatic solid tumors, LMP2, LMP2 is also known as proteasome subunit beta type 9 (PSMB9), metastatic melanoma, LMP7 is also known as proteasome subunit beta type 8 (PSMB8), metastatic melanoma, MECL1 is also known as proteasome subunit beta type 10 (PSMB10), metastatic melanoma, vascular endothelial growth factor (VEGF) in solid tumors, Kinesin spindle proteins in chronic myeloid leukemia, apoptosis suppressor B-cell CLL / lymphoma (BCL-2), nucleotide reductase M2 (RRM2) in solid tumors, Furin in solid tumors, Polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol transfer 1 (DGAT1) in hepatitis C virus infection, β-catenin, β2-adrenergic receptor in familial adenomatous polyposis, RTP801 / Redd1 in glaucoma, diabetic macular edema (DME) or age-related macular degeneration, and DNA damage-induced Also called induced transcription factor 4 protein, vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteriolaryngeal inflammatory ischemic optic neuropathy, keratin 6AN17K mutant protein in congenital pachyonychia, influenza infection with influenza A virus A genome / gene sequence in influenza, severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection, respiratory syncytial virus infection genome / gene sequence in respiratory syncytial virus infection,Ebola filamentous virus genome / gene sequence in Ebola virus infection, Hepatitis B and C virus genome / gene sequence in Hepatitis B and C virus infection, Herpes simplex virus (HSV) genome / gene sequence in HSV infection, Coxsackievirus B3 genome / gene sequence in Coxsackievirus B3 infection, silencing of pathogenic alleles (allele specific silencing) of torsin A (TOR1A) in primary myotonic disorders, pan-class I and HLA alleles in transplantation, mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP), or a transcript inhibitory nucleic acid that binds to any of the above genes or sequences.

[0127] In addition, studies have shown that heterologous nucleic acid can encode shRNA to interfere with the expression of a target gene, heterologous nucleic acid can encode gRNA and Cas9 to achieve knockout of a target gene, and heterologous nucleic acid can encode gRNA and dCas9 to achieve endogenous overexpression of a target gene.

[0128] Host cell line screening By selecting an appropriate host cell, the yield of the viral vector can be significantly improved. Preferred cells are suitable for serum-free culture and / or suspension culture. As described above, when cell culture and amplification are performed in serum-free medium, potential immunogenic substance contamination and animal-derived components in the final product are reduced, and the downstream purification process is simplified, and the simplification of the purification process can improve the yield and activity of the viral vector. In addition, suspension culture is more suitable for large-scale production of viral vectors than adherent culture.

[0129] Serum-free culture In general, the growth of all animal cells depends on the presence of serum. Most cells cannot grow without the addition of serum. Serum-containing media is usually supplemented with bovine serum.

[0130] Serum has complex components, including proteins and nutrients that aid in the transport of substances, as well as hormones and factors that promote cell growth. Due to the complexity of serum as a natural component, serum performs various functions during cell culture, including providing hormones that maintain cell growth and promote the growth of cultured cells; supplementing nutrients that are absent or present in low amounts in basal media; providing binding proteins to promote cell recognition and utilization of vitamins, lipids, and other hormones; in some cases, the binding proteins can bind to toxic metals and pyrogens and perform detoxification; providing factors that can promote cell adhesion and spread of cells on plastic culture matrices; acting as an acid-alkalinity buffer; and providing protease inhibitors to inactivate remaining trypsin when cells are digested and protect cells from injury. However, the use of bovine serum carries the risk of contamination with foreign viruses and pathogenic factors, and the biological activity and factors of different batches of bovine serum are inconsistent, resulting in low reproducibility of products and experimental results. Residual bovine serum in the product is also likely to cause allergic reactions to the serum of viral vector recipients.

[0131] Serum-free medium, abbreviated as SFM, is a cell culture medium without the addition of serum. It is the third type of culture medium following natural medium and synthetic medium. Compared with traditional culture media, serum-free medium does not contain animal serum or its biological extracts, but can maintain cells to grow and proliferate for a long time in vitro. SFM has clear components and a simple preparation process, and is widely applied in the modern biotechnology field. It is also a powerful tool for elucidating basic research problems of cell growth, proliferation, differentiation and gene expression regulation.

[0132] There are currently two types of serum-free media: one that does not contain any animal-derived additives, and one that does not contain any undefined additives. Currently, the following four are widely used:

[0133] The first type is a general serum-free medium, which is made by preparing cell culture media using various biomaterials that can replace serum functions, such as biopolymers such as bovine serum albumin, transferrin, and insulin, as well as mixed lipids and hydrolyzed proteins extracted from serum after removing proteins, etc. Its characteristic is that the medium contains a high amount of protein, but the chemical composition of the added substances is unclear and it contains a lot of animal-derived proteins.

[0134] The second is non-animal-derived media. Many companies have developed non-animal-derived media, which take into consideration the safety of recombinant drugs, and the added components in the media are not of animal origin, and the necessary proteins are derived from recombinant proteins or protein hydrolysates, and these components can ensure the needs of cell growth and proliferation.

[0135] The third is animal protein-free media. The media are completely free of animal-derived proteins, but some additives are derived from hydrolyzed fragments of plant proteins or other derivatives, such as synthetic polypeptide fragments. Such media are stable in composition, but require the addition of steroid hormones and lipid precursors, and have high specificity for cultured cells.

[0136] The fourth type is a medium with limited chemical components. This type of medium is currently the safest and most ideal medium, and can ensure consistency between batches of medium. The small amount of animal-derived protein hydrolysate and protein added to it are all clearly defined components. The characteristics of this medium are that the properties of the medium are clearly defined, and it is convenient to formulate the medium.

[0137] The advantages of serum-free medium include: avoiding the quality variation between serum batches, improving the reproducibility of cell culture and experimental results, avoiding the toxic effect of serum on cells and serum-derived contamination, avoiding the impact of serum components on experimental research, being favorable for the differentiation of in vitro cultured cells, improving the expression level of products and facilitating the purification of cell products, the components are stable and can be mass-produced, and it does not contain mitogen inhibitors and can promote cell proliferation, etc. The disadvantages of such media include that cells are affected by some mechanical and chemical factors in serum-free medium, the storage and application of the medium is not as convenient as traditional synthetic media, the cost is high, the targeting is high, one serum-free medium is only suitable for the culture of certain cells, cells at different differentiation stages of development require different formulations, and the selection of growth factors and cytokines is particularly important. At the same time as removing serum, some protective effects of serum proteins are also removed, so there are high requirements for the purity of reagents, water, and cleanliness of instruments.

[0138] Serum-free medium is formed by adding hormones, growth factors, attachment factors and binding proteins to a complete basal nutrient medium.

[0139] Early basal media used in cell culture include natural media such as plasma clot, lymph, soybean peptone, and embryo extract. In 1950, Morgan et al. developed 199 medium based on their previous research, which marked a new stage in the development of animal cell culture media and the stage of synthetic media. Synthetic media are basal media that combine a certain proportion of amino acids, vitamins, inorganic salts, glucose, etc. according to the needs of cell growth. Currently, there are more than 100 types of synthetic media on the market. Among the many synthetic media, MEM, DMEM, RPMI1640, F12, and TC100 are the most widely used. Because the components of the basal medium are completely known, some components of the basal medium can be appropriately adjusted when culturing different cell lines to better meet the nutritional requirements of the cell line or improve the expression level of the target protein.

[0140] Supplementary factors in serum-free media are also called supplementary factors, and are a general term for various factors that replace serum. Most serum-free culture media require the addition of 3 to 8 additional factors, and no single factor can replace serum. More than 100 such factors are known, including essential factors such as insulin, sodium selenite, and transferrin, and many others that act as helpers. Supplementary factors are classified into four types based on their function.

[0141] The first supplementary factors are hormones and growth factors. Many cells cultured without serum need to be supplemented with hormones such as insulin, growth hormone, glucagon, etc. Most cell lines require insulin, which is a polypeptide that can bind to insulin receptors in cells to form complexes and promote the synthesis of RNA, proteins, and fatty acids, and is an important cell survival factor. In serum-free cell culture, the concentration of insulin used is 0.1-10 μg / ml. Jan et al. believe that the rapid depletion of insulin is the main cause of the decrease in the specific cell growth rate in batch culture. In addition, thyroxine, etc., and steroid hormones such as progesterone, hydrocortisone, and estradiol are also common supplementary factors in serum-free cell culture. Different cell lines have different requirements for the type and quantity of hormones. Growth factors are supplementary factors necessary to maintain the survival, proliferation, and differentiation of cells in in vitro culture. According to their chemical properties, they can be classified into polypeptide growth factors and steroid growth factors. Growth factors added to serum-free media are mainly polypeptide growth factors, and 20 to 30 types of polypeptide growth factors have been identified in recent years, more than half of which can be obtained as recombinant growth factors by genetic recombination techniques. Growth factors commonly found in serum-free media include epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF). Growth factors are effective mitogens and can shorten the doubling time of cell populations.

[0142] The second supplementary factor is a binding protein. There are two binding proteins, one is transferrin and the other is albumin. Most mammalian cells have a specific transferrin receptor, and the binding of the receptor to a complex of transferrin and iron ions is the main source for cells to obtain iron, a trace element required by cells, and transferrin also has the properties of a growth factor and can bind to other trace elements such as vanadium. The amount of transferrin required varies depending on the cell. Albumin is also a commonly used supplementary factor in serum-free media. It stabilizes and regulates the activity of vitamins, lipids, hormones, metal ions and growth factors in serum-free media by binding to them, and also binds to toxins and reduces the effects of proteases on cells.

[0143] The third supplementary factor is an adhesion factor. Most eukaryotic cells need to be attached to a suitable substrate when growing in vitro. Cell adhesion is a complex process, including adhesion factors attaching to the surface of a container or vector, binding of cells to adhesion factors, etc. Adhesion factors commonly used in serum-free culture include components in the matrix and serum, fibronectin, collagen, laminin, polylysine, etc. In suspension culture such as the present application, there is no need to add adhesion factors to the medium.

[0144] Serum-free culture of some cell lines also needs to supplement with some low molecular weight chemicals such as trace elements, vitamins, and lipids, vitamin B is involved in cell metabolism mainly in the form of coenzymes, vitamin C and vitamin E have antioxidant effects, butanediamine and linoleic acid provide lipids required for cell membrane synthesis and water-soluble lipids required for cell growth.

[0145] Currently, serum-free media are widely applied in large-scale animal cell culture. In the application fields of biological products such as vaccine growth, mabs and various bioactive proteins, the optimization of serum-free media components can maintain high-density cultures of different cells in the most favorable environment for cell growth and expression of the desired product, and reduce production costs. In human cell culture, the application of serum-free media can also selectively control and avoid the overgrowth of fibroblasts. Under serum-free culture conditions, the growth rate of some cells and the yield of antibodies are several times higher than when serum is present.

[0146] In addition to producing biological products, serum-free media are also widely applied in the fields of cell biology, pharmacology, and oncology. For example, serum-free media can be used to study the conditions for cell differentiation. Other components of serum-free media may be completely and reliably known chemicals, and therefore the types and amounts of substances with important biological activity can be increased or decreased according to the needs of the study. This has provided an effective means for the study of conditions for cell differentiation. For example, serum-free media can be used to select target cells from a culture containing a variety of cells. By removing or removing some components in serum-free culture, the excessive growth of non-target cells in primary tissue culture can be suppressed and the purpose of selecting target cells can be achieved. Serum-free culture is also used in the study of tumor pathology and etiology. For example, it is used to study the effects of carcinogenic factors on cells, the ability of tumor cells to respond to peripheral signals that can induce terminal differentiation of normal cells, or the relationship between the growth and migration of normal and tumor cells and basement membrane signals. In addition, serum-free culture can also be used to study the interactions of cells with hormones, growth factors, drugs, etc.

[0147] Commercially available serum-free cell growth media include FreeStyle TM 293(Gibco TM , Life Technologies), DMEM / F12 (Gibco TM , Life Technologies), SFM4Transfx-293(HyClone TM, ThermoScientific), CDM4HEK293(HyClone TM , ThermoScientific), StemPro-34SFM (Gibco TM , Life Technologies), FreeStyle F17 (Gibco TM , Life Technologies), 293SFMII (Gibco TM , Life Technologies), CD293 (Gibco TM , Life Technologies), LV-MAX(A35834-01, Gibco TM ), Expi293 (Gibco TM , A14351-01), EX-CELL(R)293 (Sigma, 14571C-1000mL), BanlanCD HEK293 (Irvine, 91165), Pro293s-CDM (Lonza, 12-765Q), OPM-293 CD03 (Okuura Susumu, 81070-001), Celkey ​​CD HEK293 (Kenjun, 10804-19008) medium. As used herein, these serum-free media may be used for screening and / or culturing cells.

[0148] Suspension culture Suspension culture refers to the culture of single cells and small cell pellets by dispersing and suspending cells in the medium. It is a method of culturing non-adhesion-dependent cells. Some adhesion-dependent cells can be cultured in this way after adaptation and selection. The scale of suspension culture can be easily increased by simply increasing the volume. When the depth of the medium exceeds a certain value, the medium needs to be agitated by a shaking or rotating device. When the depth of the medium becomes too deep, carbon dioxide and oxygen need to be introduced to ensure sufficient gas exchange.

[0149] According to cell adhesion, the suspension culture technology can be divided into suspension cell culture process and adherent cell microcarrier suspension culture process. Suspension cells (CHO cells, BHK21l cells, HEK293 cells, etc.) can be produced and grown directly in the reactor, the cells grow freely, the culture environment is uniform, they can be easily sampled, the culture operation is simple and controllable, it is easy to scale up, the contamination rate and cost are low, whereas adherent cells need to use microcarriers when cultured in the reactor, the nutritional environment at the contact site between the cells and the spheres is poor, the culture, sampling observation and scale-up process are complicated, and the cost is high.

[0150] According to the culture method, the cell suspension culture process can be divided into batch culture, fed-batch culture and perfusion culture. Batch culture can intuitively reflect the growth and metabolic changes in the cell bioreactor, and is easy to operate, but there are many waste products of the initial metabolism, which inhibits cell growth and the cell growth density is not high. Fed-batch culture is easy to operate, has a high yield, is easy to scale up, and is widely used, but needs to design the fed-batch medium. Perfusion culture has a small culture volume, a large recovery volume, a short residence time of the product in the tank, and can be timely collected and stored at low temperature, which is advantageous for maintaining the activity of the product, but has problems such as complicated operation, low utilization efficiency of the cell culture medium, and easy clogging of the rotary filter. Different cell culture methods are used for different virus vectors. For example, for biological products that are secreted and have a fast decline in product activity, it is appropriate to use perfusion culture, and perfusion culture is also more suitable for microcarrier culture.

[0151] After selecting a suitable production process, process control is key. To ensure that cells grow in an optimal environment, various operating parameters must be controlled using online monitoring of the reactor. Cells are temperature sensitive, and the culture temperature must be controlled at 35°C to 37°C using appropriate heating or cooling methods to avoid cell damage. The appropriate pH range for animal cell growth is generally 6.8 to 7.3; a pH lower than 6.8 or higher than 7.3 may have a negative effect on cell growth.

[0152] Whether cell culture can be scaled up is an important premise for selecting a reactor. The scale-up of suspension culture is mainly done by increasing the reactor volume or increasing the number of reactors. Increasing the reactor volume can save a lot of installation work and labor costs, while multiple small reactors are flexible to operate, but cost is high. In overseas biological product production, it is common to use large-scale bioreactors that can be scaled up in stages. The selection and configuration of bioreactors should also consider and meet the requirements of the production process and production capacity. Reactor interface standardization, accessory supply speed, after-sales service quality, etc. are all factors to consider when selecting a large number of bioreactors to avoid production delays.

[0153] In the liquid suspension culture process, it is important to note that the culture will enter a stationary phase of division after a period of growth, so subculture the cells in a timely manner. For most suspension cultures, the cells will reach maximum density on the 18th to 25th day of culture, at which time they should be subcultured for the first time. During subculture, large cell clumps and inoculum residues should be removed.

[0154] Cell line screening The essence of cell screening acclimation is to apply modern cell biology technology and study aspects such as adaptation to different culture methods (e.g., suspension), different media (e.g., serum-free media), reduction of apoptosis rate, improvement of cell viability, extension of cell life cycle, and improvement of product concentration, to screen cell lines suitable for production. To achieve stabilization of acclimation, high-density cell culture is usually shifted to low-density cell culture, and culture with gradually reduced serum concentration is performed from high-concentration serum culture. Because it is difficult to repair after cell damage, the cell viability during acclimation must be maintained at 90% or more. The proliferation ability of cells directly affects the production ability, and it is necessary to measure their proliferation ability during acclimation. In order to avoid changes in cell expression characteristics after acclimation, it is necessary to measure their expression and secretion ability when acclimating cells. The proliferation ability, viability, and production ability of cells after acclimation are required to meet the requirements of industrial mass production. Since the nutritional needs of specific cells are different and the difficulty of achieving their habituation is also different, it is necessary to select an appropriate cell culture medium during habituation and precisely supplement some nutritional components to meet the specific needs of the cells, so that the habituated cells can maintain their suspension or serum-free growth characteristics.

[0155] In addition, in suspension culture, cells are prone to clumping. When cells clump, many of the plasmids used for vector production cannot be transfected into the cells, and the cells between the large clumps may die due to lack of nutrients. At present, the method of improving cell clumping is mainly by adding anti-clumping agents. The addition of anti-clumping agents obviously reduces the transfection efficiency, and as a result, the cells cannot transfect and package the adeno-associated virus.

[0156] There is a need in the industry for an efficient and simple method for adapting / screening cell lines that have high dispersibility, high growth density, and high viral vector production.

[0157] In the present application, the inventors have mainly improved the adaptation / screening methods for HEK293 cells, which are currently most commonly used for producing adeno-associated viruses.

[0158] The inventors believe that by not using the industry-standard culture step of gradually reducing the serum concentration from high-concentration serum culture, but instead directly adding adherent cells cultured in normal medium to serum-free medium to perform suspension culture, they can significantly save time and have high dispersibility (i.e., less aggregation), high growth density, and also contribute to some extent to the success of screening cell lines that produce high amounts of viral vectors.

[0159] The inventors of the present application further found that the use of certain specific serum-free suspension media, such as LV-MAX production medium (Gibco, A35834-01), Expi293 expression medium (Gibco, A14351-01) and OPM-293 CD03 medium (Susumu Okuura, 81070-001), is more advantageous for screening cell lines that are highly dispersible and do not aggregate. Specifically, the cells screened using these three types of media were able to grow at a high density (~1×10 7 The cell density can reach 1000 cells / ml, and the proportion of aggregated cells is low, which is advantageous for screening non-aggregated subclones. Among these three media, OPM-293 CD03 medium (Okuura Susumu, 81070-001) has the best screening effect.

[0160] The present application screened mainly HEK293 cells, which are commonly used for producing adeno-associated viral vectors, because they have rapid proliferation and high transfection efficiency, allowing efficient production of viral vectors, and have the potential to produce viral vectors on a large scale.

[0161] Specifically, the present application provides a method for screening or adapting a HEK293 cell line suitable for serum-free suspension culture, the method comprising: i) taking HEK293 cells cultured in an adherent culture in a serum-containing medium and culturing them in suspension in a serum-free medium; and ii) selecting a monoclonal cell line suitable for serum-free suspension culture.

[0162] The serum-containing medium in step i) may contain 10% serum, for example 10% fetal bovine serum.

[0163] Step i) The medium suspension culture may comprise a shaking culture at 37° C. and 8% CO2.

[0164] The suspension culture in step i) may include passaging of the cells.

[0165] The serum-free medium may be selected from LV-MAX production medium (Gibco, A35834-01), Expi293 expression medium (Gibco, A14351-01) and OPM-293 CD03 medium (Susumu Okuura, 81070-001), preferably OPM-293 CD03 medium (Susumu Okuura, 81070-001).

[0166] Step ii) may involve selecting a monoclonal cell line that is suitable for serum-free suspension culture and is highly dispersible (i.e., does not aggregate).

[0167] Step ii) may include selecting monoclonal cells by at least one round, for example two rounds, of finite dilution and photographic imaging, and culturing the selected monoclonal cells in suspension.

[0168] The suspension culture of the selected cells in step ii) may include shaking culture under conditions of 37° C. and 8% CO 2 . The suspension culture of the selected cells in step ii) may include passaging of the cells.

[0169] In the process of carrying out the method, there is no need to add any anti-agglomerating agent.

[0170] In the method of the present application, 1) cells cultured in adherent culture in serum-containing medium are directly cultured in serum-free medium, and there is no step of gradually reducing serum, which greatly shortens the adaptation and screening process, and the adaptation and screening period can be shortened to 20 days; 2) cells are cultured using serum-free medium, which avoids potential immunogenic substance contamination and animal-derived components, and simplifies the downstream purification process; 3) a serum-free medium selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium, especially OPM-293 CD03 medium, is used, which is more advantageous for screening HEK293 cell lines suitable for suspension culture, i.e., with high growth density and high dispersibility; 4) the use of anti-aggregation agents is avoided, and the transfection efficiency of cells is not affected; and 5) two rounds of finite dilution and imaging ensure the monoclonal origin, ensuring that the cells provide stable virus expression levels, while also providing intuitive data support for the monoclonal origin.

[0171] Finite dilution and imaging After suspension adaptation, the adaptability, growth rate, toxin production capacity, etc. of each HEK293 cell to such a culture method are not uniform, and as a result, the overall growth and toxin production level of the cells are not high. Monoclonal origin is an essential condition for the quality and stability of the expression level of the product, and it is necessary to screen clones that do not aggregate and have high toxin production capacity. If the selected monoclonal is not a "true" monoclonal, when the clone is used in the subsequent toxin production test experiment, the phenomenon of unstable virus expression levels of cells with different passage numbers may occur, which will have a serious impact on the quality and stability of the subsequent product.

[0172] Currently, the methods for screening monoclonal cells include: (1) performing two rounds of finite dilution and controlling the diluted cell density to less than 0.5 cells / well; (2) one round of finite dilution plus monoclonal imaging photography; (3) FACS plus one round of finite dilution or monoclonal imaging photography; and (4) semi-solid culture. The problem that exists is that the cell viability after FACS sorting is low, and HEK293 cannot be selected and photographed in semi-solid medium, and the data derived from monoclonals cannot be provided.

[0173] Currently, monoclonals are often screened using a single round of finite dilution plus monoclonal imaging.

[0174] Finite dilution is a cell cloning method commonly used in laboratories. However, because the finite dilution method may add multiple cells to one well, subcloning must be performed multiple times to improve the probability of monoclonality.

[0175] Imaging techniques offer a very intuitive method to provide supporting data, ensure the clonal origin of production cell lines, and can replace some additional laboratory work. However, several factors affect the validity (and "accuracy") of an image, e.g., calibration, focus, illumination, depth of focus, resolution, etc.

[0176] In this application, a method of adding monoclonal imaging to at least one round, for example, two rounds of finite dilution is used, which not only can provide intuitive data support for monoclonal origin, but also can compensate for the deficiencies of imaging technology to a certain extent, and greatly improve the probability of monoclonality.

[0177] Cell lines producing AVV of the present application From HEK293 cells purchased from ECACC, various cell lines with high growth density and high dispersion were obtained by the cell line screening / adaptation method of the present application.

[0178] One of them is PowerS TM It was named -293 and deposited at the China General Microorganism Collection Center (CGMCC) under the Budapest Treaty on August 9, 2021, with the deposit number CGMCC No.: 23020.

[0179] PowerS TM The -293 cell line can be grown in suspension in serum-free medium, which may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium, in particular OPM-293 CD03 medium.

[0180] PowerS TM -293 cell line does not need to appear cell clumps during the suspension culture process.

[0181] PowerS TM The -293 cell line may be used for the production of viral vectors, such as adeno-associated viruses.

[0182] PowerS TM The -293 cell line can grow in suspension culture in serum-free medium, with high proliferation density, good survival rate, stable virus productivity after subculture, no cell clumps, i.e., high dispersibility, and therefore no need to add additional anti-aggregation agents during the suspension culture process.

[0183] Screened / acclimatized PowerS for the following indicators: TM -293 cell lines were tested and all met the criteria: 1) Cell viability >90%; 2) The doubling time is 25 to 30 hours. 3) The cell density in the logarithmic phase is 3–4 × 10 6 cells / ml, 4) Maximum density > 1×10 7 cells / ml, 5) The percentage of aggregated cells is <10%, and the number of cells in each cell cluster is ≦5; 6) Adeno-associated virus packaging titer ≥ 1 x 10 11 Vg / ml, 7) PCB (the original cell bank) has completed comprehensive testing for sterility, mycoplasma, etc. 8) Cell stability is ≧50 PDL (population doubling level).

[0184] Specifically, PowerS TM -293 cells had a short doubling time of approximately 24 hours in OPM-293 CD03 medium, with a logarithmic cell density of 1.31 × 10 7 cells / ml, with a maximum density of 1.6 × 10 7 cells / ml, and the virus production capacity of the cells does not decrease with increasing number of passages (at least up to 20 passages).

[0185] Compared to commercially available virus-producing cells, PowerS TM -293 produces more viral vector particles after transfecting with viral plasmids.

[0186] The technical solutions of the present invention will be described in more detail below through the examples and with reference to the drawings. Unless otherwise specified, the methods and materials in the examples described below are all common products that are commercially available. As can be understood by those skilled in the art, the methods and materials described below are merely illustrative and should not be construed as limiting the scope of the present invention.

[0187] Example 1. Serum-free suspension adaptation of HEK293 cells HEK293 cells purchased from ECACC were incubated in a water bath at 37° C. for about 1 minute, and the cells in the cryotube were rapidly thawed until the ice in the cryotube disappeared.

[0188] The cells were transferred from the cryotube to a centrifugal tube, 4–5 ml of prewarmed DMEM (containing 10% FBS) was added, and the mixture was centrifuged at 200 g for 5 min.

[0189] The supernatant in the culture flask was discarded, and the cell pellet was resuspended in 3 ml of DMEM (containing 10% FBS). Based on the results of cell counting, the required number of cells was added to a T75 culture flask, and the T75 culture flask was placed in a 37°C, 5% CO2 incubator.

[0190] The cells were incubated for three days, and when the cell confluence reached >90%, the cell medium in the culture flask was discarded. Here, cell confluence refers to the percentage of the surface area of ​​the culture vessel covered by the monolayer cells, as observed under a microscope, for cells grown in a monolayer. The adhesion layer was washed off once with DPBS. The cells were incubated with 0.25% trypsin (company: Gibco, catalog number: 12563-029) for 2 minutes, and after the cells detached, 4-5 ml of DMEM containing 10% FBS was added to the T75 culture flask. The cells were transferred to a centrifuge tube without a centrifuge and centrifuged at 200 g for 5 minutes.

[0191] The supernatant was aspirated and the cell pellet was resuspended in 6 ml of DMEM (containing 10% FBS). Based on the results of the cell count, the required number of cells was added to a T75 culture flask, and the T75 culture flask was placed in a 37°C, 5% CO2 incubator.

[0192] Cells were passaged when they were fully adapted for adherent growth and reached >90% confluence.

[0193] A growth curve test was performed after the cells were passaged for three generations. As shown in Figure 1, in the 7-day growth curve test, the adherent cells had a doubling time of about 17 hours (less than 24 hours), and were in good growth condition, and could be used for suspension acclimatization.

[0194] At the same time, AAV2, AAV5, AAV8, and AAV9 virus packaging tests were performed on adherent cells to observe the packaging ability of adherent HEK293 cells. If the cells grow uniformly and reach confluence ≥ 90%, transfection can be performed; otherwise, the culture should be continued within 24 h and the culture time should be recorded. The medium was replaced with 9 mL of DMEM before transfection.

[0195] First, prepare transfection complex A. Specifically, add each plasmid to a 15mL centrifuge tube containing 0.5ml of transfection medium DMEM according to the numbers in Table 1 at a molar ratio of vector plasmid (pAAV-EGFP): packaging plasmid (pRC2 / pRC5 / pRC8 / pRC9): helper plasmid (pHelper) = 1:1:1, gently pipette 3-5 times to mix the plasmids evenly, and mark the centrifuge tube with A, where all vector sequences are derived from addgene and synthesized by Jinsirui.

[0196] Next, transfection complex solution B was prepared. Specifically, PEI pro was added to a 15 mL centrifuge tube containing 0.5 mL of transfection medium DMEM at a volume ratio of 2:1 (transfection reagent:DNA), i.e., 30 μl, as shown in Table 1, and mixed evenly by gently pipetting 3 to 5 times. The centrifuge tube was marked with B.

[0197] [Table 1] Solution B was added to solution A, and the mixture was mixed evenly by gently pipetting 8 to 10 times and allowed to stand for 15 minutes to prepare a transfection complex with a total volume of 1.0 mL.

[0198] After standing for 15 min, the four aliquots of the transfection complex mixture were slowly and evenly added to four T75 culture flasks at 1.0 ml per T75 flask. After 6-8 h, the mixture was replaced, i.e., the old medium in the culture flask was sucked out and replaced with 15.0 mL of DMEM containing 2% FBS. After 72 h, the virus was harvested and titered.

[0199] As shown in Figure 2, the average packaging titer of adherent cells was 2.5 × 10 10 The concentration is expressed as vg / ml (the unit means the number of copies of target genome GFP contained per ml of virus solution), and can be compared with the results of subsequent packaging of suspension-adapted monoclonal cells.

[0200] Then, LV-MAX production medium (company: Gibco, catalog number: A35834-01), Expi293 expression medium (company: Gibco, catalog number: A14351-01), and FreeStyle TM The cells were transferred to serum-free medium (SFM) including 293 expression medium (company: Gibco, catalog number: 12338-018), BanlanCD HEK293 medium (company: Irvine, catalog number: 91165), and OPM-293 CD03 medium (company: Susumu Okuura, catalog number: 81070-001) and cultured in serum-free medium. Specifically, cells that reached 85% or more confluence in a T75 culture flask were digested with 0.25% trypsin and centrifuged at 200g for 5 minutes. The supernatant was aspirated, fresh SFM was added to resuspend the cells, the cells were transferred to a 125ml shake flask, and the cells were cultured in a shaker with a shaking diameter of 19 mm and a rotation speed of 125 rpm in an incubator at 37°C and 8% CO2. The cells were cultured in a shaker with a shaking diameter of 19 mm and a rotation speed of 125 rpm at a cell density of 3–6 × 10 6 When the cell inoculation density reaches 0.35–0.55 × 10 cells / ml, subculture is performed. 6 cells / ml and were cultured for 3–4 days until viability was greater than 80%.

[0201] As shown in Figure 3, the adherent ECACC293 cells can grow normally after three generations of suspension acclimation in OPM-293 CD03, LV-MAX and Expi293 media, and the cell viability is more than 80%. However, in BanlanCD and FreeStyle media, the cells grow slowly, and the cell growth essentially stops when the second generation is cultured. Therefore, BanlanCD and FreeStyle media are not suitable for the growth of suspension HEK293 cells, and subsequent experiments were not performed. In this case, the suspension cells cultured in three types of serum-free media, OPM-293 CD03, LV-MAX and Expi293, were photographed to maintain colony morphology, and the growth state of the suspension cells in the three types of media is good, as shown in Figure 3. The suspension cells cultured in the three types of serum-free media were photographed at 0.35 x 10 6 The cells were inoculated into a cell culture flask at 1000 cells / ml and cultured for 7 days. The cell density and viability were measured every day using a cytometer (manufacturer: Beckman, model number: Vicell), and growth and viability curves were created. As shown in Figure 4, the floating cells in OPM-293 CD03 medium were in good growth condition, with a cell density of approximately 1.3 × 10 7 cells / ml and cell viability is still above 60% on day 7.

[0202] Cells that could reach higher densities by passaging were selected, specifically, cells that reached a buoyant density of up to 1.0 × 10 in OPM-293 CD03 medium. 7 A cell line was selected with a final density of 1.0 × 10 cells / ml and named HEK293 suspension cells. 7 The cells were frozen and stored until the total cell density reached 10–20 cells / ml, and the resulting serum-free suspension culture cell line was screened.

[0203] Example 2. Monoclonal derivation of serum-free suspension-adapted HEK293 and optimization of cell aggregation In order to further solve the problems of aggregation and monoclonal origin of serum-free suspension-adapted HEK293 cells, we took the cells suitable for OPM-293 CD03 medium, and performed clone screening by adding monoclonal imaging to two rounds of finite dilution, the specific steps are as follows:

[0204] First round finite dilution After harvesting the cells, the cell density was adjusted to 1 × 10 5 The cells were diluted to 1×10 cells / ml. 5 100 μl of the cell suspension was drawn up at 10 cells / ml and added to 9.9 ml of OPM-293 CD03 cell culture medium to obtain a cell density of 1 × 10 3 Adjusted to cells / ml.

[0205] Cell density is 1 x 10 3 0.5 ml of cell suspension was drawn up and placed into 100 ml of cell culture medium to adjust the cell density to 5 cells / ml. The diluted cell suspension was added to each well of a 96-well plate at 100 μl per well.

[0206] After 7 days of observation, singly grown cells were selected under a microscope; singly grown cells appeared as a single cell cluster in the 96-well, while cells that were not singly grown appeared as two or more cell clusters.

[0207] The medium was replenished every 7 days, and after 14 days the cells grew at a faster rate.

[0208] Cells grown alone were transferred to 24-well plates when they reached 50% confluence, added with 1.5 ml of fresh screening medium, and incubated stationary.

[0209] Observation began on the third day. When the cells in 2 / 3 wells reached 50% confluence, they were transferred to a 6-well plate, 1 ml of fresh screening medium was added to the 6-well plate, and the plate was cultured in a shaker at 125 rpm.

[0210] If the cells were not in good condition, they were subcultured on 6-well plates on the 6th day, and some of the high-density clones were diluted and continued to be cultured until the cells recovered, after which 1 ml of fresh screening medium was added and the cells were cultured in a shaker at 125 rpm.

[0211] When 2 / 3 wells in the 6-well plate reached 80% confluence, the cells in the 6-well plate were transferred to a 125 ml shaking flask for suspension culture and placed in a shaker, and then observed under a microscope every day. The cell growth density and condition were examined under a microscope, and clones that grew quickly and did not aggregate or aggregated lightly were screened.

[0212] The majority of cells have a density of 3 × 10 6 If the cell density exceeds 100 cells / ml and the viability is 95% or higher, transfection can be performed. Specifically, some cells were taken out of the shake flask and transfected with GFP main plasmid AAV2 (the original shake flask was continued to be subcultured) as follows, and the five most suitable monoclonal cells with high transfection titer and high dispersibility were selected based on cell dispersion and virus titer.

[0213] Specifically, serum-free suspension-adapted HEK293 cells and a control cell line (Cat#: A35347, Thermo Fisher, i.e., LV-MAX TM Virus-producing cells in a lentivirus production system (Cat#: A35684, abbreviated as VPC herein) were taken, and 0.55 × 10 6 The cells were inoculated into a 125 ml shake flask at 100 cells / ml and cultured in suspension for three days. The cell suspension was taken from the suspension cell culture shake flask, counted, and a cell density of 3 × 10 6 Transfection can only be performed when the total number of cells / mL is reached and cell viability is greater than 95%.

[0214] Place the cells in an appropriate volume of medium so that the transfection density is 3 × 10 6The cell culture flask was placed in an incubator for suspension culture.

[0215] First, prepare the transfection complex. The medium and PEIpro transfection reagent should be stored at 2-8℃, and do not need to be preheated. The transfection reagent should be gently shaken 4-5 times before use to mix thoroughly. There is no need to place it on ice and handle it as soon as possible after taking it out of the refrigerator.

[0216] The corresponding amount of plasmid was added to the medium. The mass ratio of each plasmid was 1:3:1 (vector plasmid (pAAV-EGFP): packaging plasmids (pRC2 / pRC5 / pRC8 / pRC9): helper plasmid (pHelper)), and the plasmid dose was 0.5 μg / 1 × 10 6 cells, gently pipette 4-5 times to mix evenly, and mark as tube 1-DNA, all vector sequences are from addgene.

[0217] Add 90 µl of PEIpro transfection reagent to the transfection buffer medium, mix evenly by gently pipetting 4-5 times, and mark it as tube 2-PEIpro.

[0218] Transfer the transfection buffer in tube 2 to tube 1, pipette repeatedly 8 to 10 times or more, vortex in a vortex meter as soon as possible, and leave at room temperature for 15 min.

[0219] When transfecting cells, the transfection complex was slowly added to the cell solution to be transfected, and the shake flask was gently shaken while adding the complex. After the operation was completed, the cell culture flask was directly placed in an incubator to perform suspension culture.

[0220] After 48 hours, the virus was harvested and titer detection was performed by referring to the operation steps in the instruction manual for the AVVpro(R) Titration Kit (used for real-time PCR, TAKARA, catalog number: 6233).

[0221] After the five best monoclonal cells were determined, the corresponding shake flask cells or their passages were used to carry out the packaging test of the virus AAV2 with GFP main plasmid according to the above steps. Based on the packaging test results, five best monoclonal cells were selected, which are A7, A26, A52, A62 and G1, respectively.

[0222] PCB libraries were prepared for TOP5, and 20 copies of each were frozen and stored.

[0223] Second round finite dilution The five optimal monoclonal cell aggregates determined in the first round were subjected to finite dilution again, and the finite dilution steps of the first round were repeated (i.e., the steps from cell dilution to seeding were repeated), and imaging was performed using a monoclonal cell line analyzer (Solentim, Cell Metric) on days 0, 1, 2, and 7 of cell growth to screen the monoclonal cells, as shown in Figure 5. After 3 to 5 passages in shake flask culture, observation and photography were performed under a microscope, and clones that did not aggregate or aggregated lightly were screened, as shown in Figure 6, which were clones 1, 4, 6, 7, 8, 10, 13, 20, and 29, respectively, where clone 13 had the highest dispersibility. After subculture of the screened clones, some cells were taken out of the shake flask and transfected with the GFP main plasmid AAV2 (the original shake flask was continued to be subcultured). The results of the transfection titer are shown in Figure 8, and the titers of the screened clones transfected with the AAV2 virus were all higher than those of the control cell line. Three optimal clones with high transfection titers were selected, namely clones 13, 20 and 29.

[0224] Three monoclonal cells, clones 13, 20, and 29, were subjected to 7-day growth and viability curves in OPM-293 CD03 medium. As shown in FIG. 7 and Table 2, the three cells were in good growth condition, and the cell density of clone 13 was 1.5×10 on day 7. 7 The doubling time of clone 13 was the shortest, and the cell viability of the three cell clones was above 85%. The three cell clones were tested for AA5, AAV8, and AAV9 packaging, and the results showed that clone 13 had the highest titer when packaging the three serotype viruses, with an average value of about 1.0×10 11 vg / ml, which was clearly higher than that of the control cell line, as shown in FIG.

[0225] [Table 2]

[0226] Example 3. Passage stability experiment of clone 13 In vitro cultured cells have a high probability of gene mutation and epigenetic changes, which may be affected by culture conditions. For example, some types of cells will differentiate when conditions such as cell density, oxygen gas or carbon dioxide concentration change. As a result, during the production of biological products, these cells will have a decreased cell production capacity with an increased number of passages, and the quality of the product will be unstable between lots over time. Therefore, it is necessary to verify the stability of such cell lines in the biological production process. The inventors used AAV5 virus packaging to investigate the toxin production stability of clone 13. The specific steps are as follows: Clone 13 was cryopreserved at the 5th, 10th, 15th, and 20th passages during passage 13, respectively. After completion of cryopreservation at the 20th passage, the cryopreserved clones at the 5th, 10th, 15th, and 20th passages were incubated in a 37°C water bath for about 1 minute to rapidly thaw the cells in the cryotubes until there was no ice left in the cryotubes. Then, the cells were transferred to a 0.55 × 10 6 The cells were inoculated into a 125 ml shake flask at 100 cells / ml and cultured in suspension for three days. The cell suspension was taken from the suspension cell culture shake flask, counted, and a cell density of 3 × 10 6 Transfection can only be performed when the total number of cells / mL is reached and cell viability is greater than 95%.

[0227] Place the cells in an appropriate volume of OPM-293 CD03 medium to achieve a transfection density of 3 × 10 6 The cell culture flask was placed in an incubator for suspension culture.

[0228] First, prepare the transfection complex. OPM-293 CD03 medium and PEIpro transfection reagent should be stored at 2-8℃, and do not need to be preheated. The transfection reagent should be gently shaken 4-5 times before use to mix thoroughly. There is no need to place it on ice and handle it as soon as possible after taking it out of the refrigerator.

[0229] The corresponding amount of plasmid was added to the medium. Each plasmid was added at a mass ratio of vector plasmid (pAAV-EGFP):packaging plasmid (pRC2 / pRC5 / pRC8 / pRC9):helper plasmid (pHelper) = 1:3:1, and the plasmid dose was 0.5 μg / 1 × 10 6 cells, gently pipette 4-5 times to mix evenly, and mark as tube 1-DNA, all vector sequences are from addgene.

[0230] Add 90 µl of PEIpro transfection reagent to the transfection buffer medium, mix evenly by gently pipetting 4-5 times, and mark it as tube 2-PEIpro.

[0231] Transfer the transfection buffer in tube 2 to tube 1, pipette repeatedly 8 to 10 times or more, vortex in a vortex meter as soon as possible, and leave at room temperature for 15 min.

[0232] When transfecting cells, the transfection complex was slowly added to the cell solution to be transfected, and the shake flask was gently shaken while adding the complex. After the operation was completed, the cell culture flask was directly placed in an incubator to perform suspension culture.

[0233] After 48 hours, the virus was harvested and titer detection was performed by referring to the operation steps of the instruction manual for the AAVpro(R) Titration Kit (used for real-time PCR, TAKARA, catalog number: 6233). The detection results are shown in Figure 10, which shows that the ability of clone 13 to produce AAV5 virus did not decrease with increasing number of passages, and the AAV virus packaging ability of clone 13 was stable within 20 generations (including the 20th generation).

[0234] In summary, we finally determined that clone 13 was the optimal clone, and the clone 13 cell line was named PowerS-293 and subsequently deposited in the China General Microorganism Collection Center (CGMCC) with the number 23020.

[0235] The embodiments of the present invention are not limited to the above embodiments, and those skilled in the art may make various changes and improvements to the present invention in form and detail without departing from the spirit or scope of the present invention, all of which are deemed to fall within the protection scope of the present invention.

Claims

1. Novel human embryonic kidney HEK293 cells, deposited at the China Center for Common Microorganisms Species Collection and Management, with accession number CGMCC NO.: 23020.

2. A method for screening HEK293 cell lines suitable for serum-free suspension culture, comprising: i) removing HEK293 cells cultured in an adherent culture medium containing serum and culturing them in suspension in a serum-free medium, wherein the serum-free medium is selected from OPM-293 CD03 medium, LV-MAX production medium, and Expi293 expression medium; and ii) selecting a monoclonal cell line suitable for suspension culture.

3. 3. The method of claim 2, wherein the serum-containing medium in step i) contains 10% serum.

4. The suspension culture in step i) was incubated at 37°C and 8% CO ２ The method of claim 2, comprising shaking culture under the conditions:

5. The method of claim 2, wherein the suspension culture in step i) comprises passaging the cells.

6. 3. The method of claim 2, wherein step ii) comprises selecting monoclonal cells by at least one round of finite dilution and photographic imaging, and culturing the selected monoclonal cells in suspension.

7. The method of claim 6, wherein in step ii) the suspension culture of the selected cells comprises passaging the cells.

8. 3. The method of claim 2, wherein the serum-free medium is OPM-293 CD03 medium.

9. 3. The method of claim 2, wherein the method is carried out without the addition of an anti-agglomerating agent.

10. A cell line screened by the method according to any one of claims 2 to 9.

11. The cell line of claim 10, which does not aggregate in liquid culture.

12. Use of a medium selected from LV-MAX production medium, Expi293 expression medium, and OPM-293 CD03 medium in screening HEK293 cell lines suitable for suspension culture.

13. A method for producing a viral vector using the cell line of claim 1, comprising: i) culturing said cell line in suspension; ii) introducing a viral vector expression system into said cell line; iii) culturing the cell line under conditions in which the viral vector is produced; and iv) harvesting the viral vector.

14. The method of claim 13, further comprising the step of v) performing titer detection on the viral vector after step iv).

15. The method of claim 13 , wherein the viral vector is an adeno-associated viral vector.

16. The method of claim 15, wherein the viral vector expression system comprises a vector plasmid containing the heterologous nucleic acid, a packaging plasmid containing rep-cap, and a helper plasmid.

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