Highly dispersive HEK293T cell line and screening method thereof

Abstract

The present invention relates to a highly dispersible HEK293T cell line and a screening method thereof. Specifically, the present invention relates to a screening method for HEK293T cell lines suitable for serum-free suspension culture, cell lines screened using the method, and a method for producing a viral vector using the cell line. TIFF2024504914000003.tif68170

Description

[Technical field]

[0001] RELATED APPLICATIONS AND INCORPORATION BY REFERENCE All documents cited or referred to in this application, including but not limited to all publications, patents, and patent applications disclosed herein ("Application Cited Documents"), all documents cited or referred to in the Application Cited Documents, and manufacturer's manuals, specifications, product specifications, and product pages for any products mentioned in this application or in any Application Cited Documents, are all incorporated by reference into this application and may be used in the practice of the invention. More specifically, all references are all incorporated by reference into this application to the same extent as each individual document is incorporated by reference. Any Genbank sequences referred to herein are incorporated by reference into this application.

[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 a HEK293T cell line suitable for serum-free suspension culture, particularly suitable for serum-free suspension culture and having high dispersibility, and a HEK293T cell line screened using the method, particularly a PowerS TM The present application relates to the screened cell lines, particularly PowerS TM The present invention further relates to a method for producing a viral vector using H.293T. [Background technology]

[0004] Gene therapy and cell therapy have been developing rapidly and 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% to 80% of treatment regimens are achieved by viral vectors. Viral vectors are the key vectors that realize the delivery of therapeutic foreign genes to target sites, and they may be directly injected as drugs or used as important raw materials for drug production. At present, viral vectors for introducing foreign genes into target cells are mainly derived from viral vectors such as retroviruses, adenoviruses and adeno-associated viruses. Lentiviruses are one of the retroviral vectors and are efficient vectors that have been developed on the basis of human immunodeficiency virus type I (HIV-1). Usually, there are two methods for producing lentiviral vectors: using cell lines that stably express lentiviral packaging system elements and using transient transfection methods. The transient transfection packaging method remains the most common method for producing lentiviral vectors because it is easy to operate and time-saving, and many transgenic and messenger glycoproteins can be easily substituted in the transient transfection vector system.

[0005] HEK293 cells, also known as human embryonic kidney cells 293, are the most commonly used cells for the production of retroviruses and other viral vectors. Such cells are derived from the cell line of human embryonic kidney cells and have characteristics such as high transfection efficiency and easy culture. When the SV40 large T antigen gene is transfected into HEK293 cells, the HEK293T cell line is formed. HEK293T cells can significantly amplify plasmids containing the SV40 replication origin therein and promote protein expression, and are therefore used for the expression of various genes and the production of proteins.

[0006] Currently, most lentiviruses are produced by culturing HEK293 derived cell lines (e.g., 293T and 293E) in adherent culture in a square bottle, flat plate, or other system with the addition of fetal bovine serum, and obtaining viruses through multi-plasmid transient transfection packaging. For cell and gene therapy development companies, the demand for viral vectors during preclinical research, IND declaration, and clinical trials is limited, so lentivirus vectors are mostly produced by the method of transient transfection of adherent cells HEK293T. The adherent culture of HEK293T cells is usually carried out in the form of a cell factory, and this production method mainly increases the cell amount by increasing the number of culture units. Therefore, if more viral vectors are required, a lot of space needs to be occupied and the production labor is also great.

[0007] One way to obtain a large growth area and reduce the amount of work is to adapt the cell line producing the lentivirus to suspension culture. Compared to adherent culture, suspension culture can significantly improve the cell density. In addition, the cells may be cultured using serum-free medium, which reduces potential immunogenic contamination and animal-derived components in the final product and simplifies the downstream purification process, thereby greatly promoting the conversion of the product to clinical levels. Scaling up suspension culture can also reduce the difference between batches compared to traditional adherent culture due to the increase in culture units. However, there is a problem in the serum-free suspension adaptation process of HEK293T cells that the cells are prone to clumping. When the 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. Currently, the method of improving cell clumping is mainly by adding anti-clumping agents. The addition of anti-clumping agents significantly reduces the transfection efficiency, resulting in the cells being unable to be transfected with packaging lentivirus. Summary of the Invention

[0008] Through numerous experiments, the inventors of the present application have discovered a method for screening HEK293T cell lines suitable for serum-free suspension culture, which allows for screening of cell lines with high dispersion and high growth density.

[0009] Thus, in one aspect, the present application provides a method for screening HEK293T cell lines suitable for serum-free suspension culture, said method comprising: i) taking HEK293T cells cultured in an adherent culture medium containing serum, and placing them in a serum-free medium to culture them in suspension; and ii) selecting a monoclonal cell line suitable for serum-free suspension culture.

[0010] The serum-containing medium in step i) may contain 10% serum, for example 10% fetal bovine serum.

[0011] The suspension culture in step i) may comprise a shaking culture at 37° C. and 8% CO 2 .

[0012] The suspension culture in step i) may include passaging of the cells.

[0013] The serum-free medium may be selected from LV-MAX Production Medium (Gibco, A35834-01), Expi293 Expression Medium (Gibco, A14351-01), FreeStyle™ 293 Expression Medium (Gibco, 12338-018), CD293 AGT Medium (Gibco, 11913-019), EX-CELL® 293 Serum-Free Medium (Sigma, 14571C-1000mL), Banlan CD HEK293 Medium (Irvine, 91165), Pro293s-CDM Serum-Free Medium (Lonza, 12-765Q), OPM-293 CD03 Medium (Okuura Susumu, 81070-001) and Celkey ​​CD HEK293 Medium (Kenjun, 10804-19008). The serum-free medium is preferably LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0014] Step ii) may involve selecting a non-aggregating monoclonal cell line suitable for serum-free suspension culture.

[0015] Step ii) may comprise culturing cells in a semi-solid medium, selecting single cells from the semi-solid medium, and culturing the selected cells in suspension.

[0016] The suspension culture of the selected cells in step ii) may include shaking culture under conditions of 37° C. and 8% CO2. The suspension culture of the selected cells in step ii) may include passaging the cells, for example, three or more passagings.

[0017] The semi-solid medium may be a methylcellulose-based medium supplemented with screening medium.

[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 adherent culture in serum-containing medium are directly cultured in serum-free medium, and there is no need to gradually reduce the amount of serum, thereby shortening the screening process; 2) cells are cultured in 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 is used, which is more advantageous for screening HEK293T 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) the use of semi-solid medium facilitates the screening of monoclonals that meet the conditions.

[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 HEK293T cell lines with high dispersion and high growth density.

[0021] In another aspect, the present application relates to a HEK293T cell line screened by the above method.

[0022] In particular, the present application relates to a novel human embryonic kidney HEK293T cell line PowerS, which was deposited with the China General Microorganism Collection Center (CGMCC) under the Budapest Treaty on December 7, 2020 and bears the deposit number CGMCC No.: 21100. TM -293T is provided.

[0023] The cell line is capable of growing in suspension culture in serum-free medium, which may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0024] The cell line may be free of cell clumps during the suspension culture process.

[0025] The cell line can be used for the production of viral vectors.

[0026] PowerS TM The 293T cell line can grow in suspension culture in serum-free medium, with high growth density, good vitality, and no cell clumps, i.e., high dispersibility. Therefore, there is no need to add additional anti-aggregation agents in the suspension culture process. Compared with Gibco's CTS trademarked virus production cells (publication number: MAN0018590), PowerS TM -293T cells have a shorter doubling time and produce more viral vectors after transfection with viral plasmids.

[0027] In yet another aspect, the present application relates to the use of the screened HEK293T cell line in the production of viral vectors.

[0028] Specifically, this application relates to PowerS TMThe present application also provides a method for producing a viral vector using a cell line screened according to the present application, including 293T, the method comprising: i) culturing the cell line; ii) introducing a viral vector expression system into the cell line; iii) culturing the cell line under conditions in which the viral vector was produced; and vi) harvesting the viral vector.

[0029] The method may further comprise performing titer detection on the viral vector after step vi).

[0030] 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., 8% CO. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0031] Step i) may further comprise passaging the cell line.

[0032] 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.

[0033] The viral vector expression system may include a packaging nucleic acid vector and an envelope nucleic acid vector. The viral vector expression system may further include a helper nucleic acid vector. The viral vector expression system may further include a nucleic acid vector comprising a heterologous nucleic acid, for example a transfer nucleic acid vector comprising a heterologous nucleic acid. These nucleic acid vectors may be plasmids.

[0034] The length of the heterologous nucleic acid is 4 kb or less. The heterologous nucleic acid can encode a protein such as a polypeptide or chimeric antigen receptor (CAR), or a nucleic acid such as an siRNA. In one embodiment, the heterologous nucleic acid can encode a CAR, for example, a CAR that targets CD19, CD20, CD22, CD30, CD33, or CD269 (BCMA). In one embodiment, the CAR encoded by the heterologous nucleic acid can target an antigen involved in growth and differentiation signaling, including carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR, HER2), tyrosine kinase receptor (EPHA2), and the like. In one embodiment, the CAR encoded by the heterologous nucleic acid can target an antigen involved in tumor vascular development, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and the like. In one embodiment, the CAR encoded by the heterologous nucleic acid can target tumor stroma and extracellular matrix antigens of tumor supporting structures, including fibroblast activation protein (FAP), tenascin protein, and the like. Plasmids containing heterologous nucleic acid may include pCDH and pCMV, among others.

[0035] The viral vector may be a retroviral vector, an adenoviral vector, or an adeno-associated viral vector. The retroviral vector may be a lentiviral vector, an alpha-retroviral vector, a gamma-retroviral vector, or a foamy retroviral vector. In one embodiment, the retroviral vector is a lentiviral vector selected from HIV-1, HIV-2, SIV, FIN, EIAV, and VISNA. In one embodiment, the lentiviral vector may be an HIV-1 vector. In one embodiment, the viral vector is an adeno-associated viral vector. In one embodiment, the adeno-associated viral vector is selected from AAV2, AAV5, AAV8, AAV9, and AAV10.

[0036] The retroviral vector expression system may include a vector containing a retroviral structural protein gag-pol gene and a nucleic acid vector containing the env gene or a functional substitute thereof. The env gene or a functional substitute thereof may encode the vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof. The retroviral vector expression system may further include a nucleic acid vector containing the helper gene rev or a gene similar thereto. The retroviral vector expression system may further include a nucleic acid vector containing a heterologous nucleic acid, for example a transfer nucleic acid vector containing a heterologous nucleic acid.

[0037] In one embodiment, a method is provided for producing a lentiviral vector, comprising introducing into a cell line three nucleic acid vectors, each comprising the HIV-1 gag-pol-rev genes, a nucleic acid encoding vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof, and a heterologous nucleic acid.

[0038] In one embodiment, a method for producing a lentiviral vector includes introducing into a cell line four nucleic acid vectors, each of which includes an HIV-1 gag-pol gene, an HIV-1 rev gene, a nucleic acid encoding vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof, and a heterologous nucleic acid. The nucleic acid vector including the heterologous nucleic acid may include a self-inactivating lentiviral long terminal repeat.

[0039] Step iii) 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., 8% CO. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0040] Step iii) may involve passaging the cells.

[0041] 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.

[0042] 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."

[0043] 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.

[0044] 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]

[0045] 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] The morphology of cells acclimatized in different media is shown. [Diagram 2] Growth curves of cells acclimatized in three serum-free media are shown. [Diagram 3] The morphology of monoclonal cells adapted in three serum-free media is shown. [Figure 4] The cell morphology (A) and growth curve (B) of the cell line PowerSTM-293T are shown.

[0046] Description of the deposit: The novel human embryonic kidney HEK293T cell line PowerS TM -293T was deposited at the China General Microorganism Collection Center (CGMCC) under the Budapest Treaty on December 7, 2020, with the deposit number CGMCC No.: 21100.

[0047] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] 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.

[0049] 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, high growth density, and high titer viral particle vectors, particularly retroviral vectors, especially lentiviral vector particles.

[0050] Viral Vectors

[0051] 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 the foreign genes without causing disease in the organism. In this application, "heterologous nucleic acid", "heterologous gene", "foreign nucleic acid" or "foreign gene" refers to a nucleic acid or gene that is not naturally present in a virus. Generally, a heterologous nucleic acid or foreign gene includes an open reading frame that encodes a target protein or a non-translated RNA.

[0052] The terms "viral vector," "viral particle," or "viral vector particle" are used interchangeably herein.

[0053] Known viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viruses, and the like.

[0054] Retroviruses

[0055] Retroviral vectors are the most widely used viral vectors in human gene therapy research. Retroviruses are a family of viruses that contain a pseudodiploid single-stranded RNA genome and encode a reverse transcriptase enzyme that produces DNA from the RNA genome, which can then be inserted into the host cell DNA. All retroviruses contain at least three genes, gag, pol, and env, that code for structural proteins and enzymes (reverse transcriptase, RNase H, integrase, and protease) necessary for viral replication.

[0056] To better understand retroviruses, we first describe the nucleic acid sequences shared by all retroviruses as follows.

[0057] Long terminal repeat (LTR): The basic structure of the retroviral genome includes 5'-LTR and 3'-LTR, and the genes necessary for the production of retrovirus are located between or within them. LTRs are necessary for retroviral integration and transcription. As promoter sequences, they can control the expression of retroviral genes (i.e., they are cis-acting genes). LTRs are composed of three subregions, U3, R, and U5: U3 is derived from a unique sequence at the 3' end of the RNA, R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a unique sequence at the 5' end of the RNA. To solve the safety problems associated with the generation of replication-competent viruses, a self-inactivating (SIN) vector was developed by deleting a segment in the U3 region of the 3'LTR, which contains a TATA cassette and binding sites for the transcription factors Sp1 and NF-κB (Miyoshi H et al., (1998) Development of a self-inactivating lentivirus vector. J Virol. 72(10):8150-8157). After reverse transcription and integration into infected cells, the deletion is transferred to the 5'LTR, causing inactivation of LTR transcription.

[0058] ψ: Encapsidation of retroviral RNA occurs via the ψ (psi) sequence located at the 5' end of the retroviral genome. It is well known in the art that sequences extending downstream of the psi sequence into the gag coding region are involved in the production of efficient retroviral vectors (Yan Cui et al., (1999) Contribution of viral splice sites and cis-regulatory elements to lentivirus vector function. J. Virol. 73(7):6171-6176).

[0059] Primer binding site (PBS): Retroviral genomes contain a PBS located after the U5 region of the 5'-LTR, which contains the tRNA primer required to initiate reverse transcription.

[0060] PPT: Retroviral genomes contain short stretches of purines called polypurine tracts (PPTs) near the 3' end of the retroviral genome. These PPTs function as RNA primers for plus-strand DNA synthesis in the reverse transcription process. Complex retroviruses (e.g., HIV-1) contain a second, more centrally located PPT (i.e., central polypurine tract (cPPT)), which provides a second site for initiating DNA synthesis. Retroviral vectors encoding cPPTs have been shown to have enhanced transduction and transgenic expression (Barry SC et al., (2001) Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression. Hum Gene Ther. 12(9):1103-1108).

[0061] The genomic structure of the non-coding region is well known to those skilled in the art. For example, details on the genomic structure of the non-coding region in HIV-1 can be found in NCBI Genbank Genome Accession No. AF033819, or Genome Accession No. K03455 for HIV-1HXB2 (a commonly used HIV-1 reference strain).

[0062] Gag / pol: Expression of the gag and pol genes depends on a translational frameshift between gag and pol, both of which are polyproteins that are cleaved during the maturation process. The main structural matrix of retroviral vectors, the capsid and nucleocapsid proteins, are encoded by gag. The pol gene encodes enzymes involved in viral replication, including i) reverse transcriptase, which is responsible for reverse transcription of the retroviral RNA genome into double-stranded DNA, ii) integrase, which integrates the retroviral DNA genome into the host cell chromosome, and iii) protease, which cleaves the synthesized polyprotein to produce mature and functional retroviral proteins. In one embodiment, the retroviral nucleic acid sequence encoding the gag and pol proteins is derived from the HIV-1 HXB2 sequence, which can be obtained from Genome Accession No. K03455, e.g., from base pairs 790-5105.

[0063] Env: The env ("envelope") gene encodes the surface and transmembrane components of the retroviral envelope (e.g., glycoproteins gp120 and gp41 of HIV-1) and is involved in retrovirus-cell membrane fusion. To broaden the tissue transfection potential of retroviral vectors, retroviral vectors may be pseudotyped with envelope proteins from other viruses. Pseudotyping refers to modifying the glycoprotein (GP) in the retroviral vector particle to broaden or modify the host cell range of retroviral vectors (including lentiviral vectors), for example, by using GPs obtained or derived from other enveloped viruses or by using synthetic / artificial GPs. Vesicular stomatitis virus GP (VSVg) has become the most common glycoprotein for pseudotyping retroviral vectors due to its broad transfection potential and high vector particle stability. However, one of skill in the art will appreciate that other glycoproteins can be used for pseudotyping (Cronin J et al., (2005) Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther. 5(4):387-398). The choice of virus for pseudotyping may further depend on the type of cell and / or organ to be targeted.The env protein or a functional substitute thereof may be obtained or derived from a virus selected from the following: vesiculovirus (e.g., vesicular stomatitis virus), lyssavirus (e.g., rabies virus, Mokola virus), arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), alphavirus (e.g., Ross River virus (RRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus), filovirus (e.g., Reston Ebola virus, Zaire Ebola virus, Lassa fever virus), alpharetrovirus (e.g., avian leukosis virus (ALV)), betaretrovirus (e.g., Jaagsiekte sheep retrovirus (JSRV)), gammaretrovirus (e.g., Moloney murine leukemia virus (MLV), gibbon ape leukemia virus (GALV)), ), feline endogenous retrovirus (RD114), delta retrovirus (e.g., human T-lymphotropic virus 1 (HTLV-1)), spumavirus (e.g., human foamy virus), lentivirus (e.g., Maedi-visna virus (MVV)), coronavirus (e.g., SARS-CoV), respirovirus (e.g., Sendai virus, respiratory syncytial virus (RSV)), hepacivirus (e.g., Hepatitis C virus (HCV)), influenza virus (e.g., influenza virus A), and nuclear polyhedrosis virus (e.g., Autographa californica polyhedrosis virus (AcMNPV)). In one embodiment, the env protein or a functional substitute thereof is obtained or derived from a vesicular stomatitis virus. In such an embodiment, the vesicular stomatitis virus glycoprotein (VSVg) protein may be used, which allows the retroviral particle to infect a broader host cell range and reduces the chance of recombination to produce wild-type envelope protein. In another embodiment, the retroviral nucleic acid sequence encoding the env protein or a functional substitute thereof is derived from a sequence obtainable from, for example, base pairs 3071-4720 of Genome Accession No. J02428.1.

[0064] The structural genes described herein are common to all retroviruses. Other helper genes can be present in different types of retroviruses. For example, lentiviruses such as HIV-1 contain six additional helper genes called rev, vif, vpu, vpr, nef and tat. Other retroviruses may have helper genes similar to those described herein, but they are not always referred to by the same names in the literature. For example, Tomonaga and Mikami have described various retroviral helper genes (Tomonaga K, Mikami T. (1996) Molecular biology of the feline immunodeficiency virus auxiliary genes. J Gen Virol 77(Pt 8): 1611-1621).

[0065] Rev: The helper gene rev ("regulator of virions") encodes a helper protein that binds to the Rev response element (RRE) and facilitates the transport of retroviral transcripts. The protein product of the gene allows retroviral mRNA fragments containing the Rev response element (RRE) to be transported from the cell nucleus to the cytoplasm. The RRE sequence is predicted to form a complex folded structure. This special role of rev reflects the tight coupling of the splicing and nuclear transport steps. The RRE sequence may be derived from the HIV-1 HXB2 sequence, which can be obtained from, for example, base pairs 7622-8479 or 7769-8146, particularly base pairs 7622-8479, of Genome Accession No. K03455.

[0066] Rev binds to the RRE and facilitates the transport of single-spliced ​​(env, vif, vpr, and vpu) or unspliced ​​(gag, pol, and genomic RNA) viral transcripts, resulting in downstream events such as gene translation and packaging (Suhasini M, Reddy TR. (2009) Cellular proteins and HIV-1 Rev function. Curr HIV Res. 7(1):91-100). In one embodiment, the viral expression system includes the helper gene rev or a similar gene (i.e., from another retrovirus or functionally similar system). Inclusion of the rev gene ensured efficient transport of RNA transcripts of the retroviral vector genome from the cell nucleus to the cytoplasm, especially when an RRE element is also included in the transported transcript. The rev gene may have at least 60% sequence identity, e.g., at least 70% sequence identity, to base pairs 970-1320 of Genome Accession No. M11840 (i.e., HIV-1 Clone 12 cDNA, HIVPCV12 locus). In optional embodiments, the rev gene has at least 60% sequence identity, e.g., at least 70%, 80%, 90% or 100% sequence identity, to base pairs 5970-6040 and 8379-8653 of Genome Accession No. K03455.1 (i.e., human immunodeficiency virus type 1, HXB2).

[0067] Helper genes are believed to play a role in retroviral replication and pathogenesis, and therefore many current viral vector production systems do not include some of these genes. The commonly present rev is an exception, and a system similar to the rev / RRE system can be used. In one embodiment, the nucleic acid sequence in the viral vector that can code for one or more of the helper genes vpr, vif, vpu, tat, and nef or similar helper genes can be deleted from the RNA genome of the retroviral vector particle or disrupted so that the helper genes cannot code for functional helper proteins. In another embodiment, at least two or more, three or more, four or more, or all of the helper genes vpr, vif, vpu, tat, and nef or similar helper genes are deleted from the RNA genome of the retroviral vector particle or disrupted so that the helper genes cannot code for functional helper proteins. Removal of a functional helper gene does not require removal of the entire gene, but may involve removal of a portion of the gene or disruption of the gene.

[0068] It should be understood that the nucleic acid sequence encoding the replication-defective retroviral vector particle may be the same as or derived from the wild-type gene of the retrovirus on which the retroviral vector particle is based, i.e., the sequence may be a genetically or otherwise modified version of the sequence contained in the wild-type virus. Thus, the retroviral gene introduced into the nucleic acid vector or host cell genome may refer to a codon-optimized version of the wild-type gene.

[0069] Lentiviral Vectors

[0070] As one of the retroviral vectors, lentiviral vectors are gene therapy vectors developed based on HIV-1, which can introduce target genes into animal and human primary cells or cell lines. The lentiviral genome is a plus-stranded RNA, and when the genome enters a cell, it is inverted into DNA by the antitranscriptase carried by the lentiviral genome in the cytoplasm to form a DNA preintegration complex, and after entering the cell nucleus, the DNA is integrated into the cell genome. The integrated DNA transcribes mRNA and returns to the cytoplasm to express the target protein. The expression of lentivirus-mediated genes is persistent and stable because the target gene is integrated into the host cell genome and divides with the division of the cell genome. In addition, lentiviruses can effectively infect and integrate into non-dividing cells. Due to the above characteristics, lentiviral vectors have special advantages over other viral vectors, such as adenoviral vectors that do not integrate, adeno-associated viral vectors that have a low integration rate, and traditional retroviral vectors that only integrate into dividing cells.

[0071] Lentiviral vectors are based on the lentivirus genome, and have biological safety by removing several sequence structures related to viral activity. Then, the target gene sequence and expression structure required for clinical or research are introduced into this genome backbone to create a vector. Current lentiviral vectors contain all the genetic information required for packaging, transfection, and stable integration, and are the main components of the lentiviral vector system. Lentiviral vectors carrying foreign genes can be transformed into infectious viral particles through viral packaging with the help of lentiviral packaging plasmids and cell systems, which can be collected and concentrated and then directly infected with host cells or animal models to realize the expression of foreign genes in cells or biological tissues. Research has revealed that lentiviral vectors can effectively infect various types of cells, such as nerve cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells, and mediate long-term expression of target genes. Currently, lentiviruses are also widely used in research on expressing RNAi. Lentiviral vectors can produce high-titer lentivirus expressing shRNA and express shRNA in cycling and non-cycling cells, stem cells, fertilized eggs and differentiated progeny cells, achieving specific and stable functional silencing of gene expression in various types of cells and transgenic mice, providing the possibility to rapidly and efficiently study gene function in primary human and animal cell tissues and to produce animals with reduced expression of specific genes.

[0072] Early lentiviral vectors were obtained by modifying HIV, and include HIV-1 and HIV-2 vector systems. HIV-1 is a double-stranded RNA virus whose genome structure is similar to other retroviruses and contains genes that can code for three major viral structural proteins, namely gag, pol, and env. The gag gene codes for viral core proteins, such as nucleocapsid protein (p7), inner membrane protein (p17), and capsid protein (p24), the pol gene codes for enzymes involved in viral replication, and the env gene codes for viral envelope glycoproteins, which determine the targeting of the virus-infected host. In addition, the viral genome further contains two regulatory genes, tat and rev, of which rev is mainly involved in the expression level regulation of proteins, and the protein encoded by it can regulate the expression levels of gag, pol, and env, and the protein encoded by tat is involved in the control of RNA transcription and binds to the viral long terminal repeat (LTR) to promote the transcription of all viral genes. The genome also contains four helper genes, vif, vpr, vpu, and nef. The HIV-1 genome structure also contains other sequence structures necessary for the viral life cycle, such as signals necessary for viral replication and packaging, for example, long terminal repeats at both ends of the genome, which contain cis-acting elements necessary for replication.

[0073] The establishment of HIV-1 lentiviral vector system has undergone a stepwise improvement process to gradually improve the biological safety of the vector.

[0074] The first generation HIV-1 lentiviral vector is a two-plasmid system that contains three structural genes, env, gag and pol, two regulatory genes, tat and rev, and four helper genes, vpr, vif, vpu and nef, with long terminal repeats (LTRs) at both ends, and a viral packaging signal sequence between the 5'LTR and gag. The initial packaging of lentiviral vectors is achieved by removing the trans-acting protein gene sequence in the HIV genome, and then packaging a nucleic acid vector containing the target gene and a packaging plasmid that can provide the proteins required for viral particles in trans, and co-transfecting them into host cells (e.g., 293T cells). This is a replication-defective vector system, which produces low titers of virus, can only infect CD4+ cells, and has the potential for recombination of the packaged protein DNA and vector DNA during the transfection process, as well as the safety risk of producing a replication-competent viral system.

[0075] The second generation vector is a three-plasmid expression system, in which the cis-acting sequences required for packaging, reverse transcription, and integration in the HIV-1 genome are isolated from the sequences encoding the trans-acting proteins, and then cloned into three separate plasmids, one of which is a packaging plasmid, which contains the CMV promoter and controls the expression of the complete viral structural genes except env, and uses the polyA of the human insulin gene as an addition signal instead of the 3'LTR, the second is an envelope plasmid, which contains the sequence expressing the vesicular stomatitis virus glycoprotein (VSV-G) gene, which is intended to replace the proviral env gene, and the product of this gene can expand the host range of the virus, and the third is a vector plasmid, in which the gene sequences of interest to the researcher and all the associated cis-acting elements are contained. However, this three-plasmid system retains the accessory genes of HIV-1, and this plasmid system is considered to be less safe because of the high possibility of accidental emergence of active virus.

[0076] The third generation plasmid system has removed all helper gene sequences of the HIV virus, and the removal of these accessory genes does not affect the viral titer and transfection capacity, while improving the safety of the vector.

[0077] The fourth generation vector is a four-plasmid system, which is also a lentiviral vector system that is currently widely used. Based on the third generation, a self-inactivating lentiviral vector is constructed, that is, the 3' LTR of the U3 region is deleted, making the vector lose the HIV-1 enhancer and promoter sequences, and RNA cannot be transcribed even in the presence of all viral proteins, and the env gene is placed on a single expression plasmid, the tat gene is removed, and a heterologous promoter sequence is substituted. Thus, four plasmid systems, namely pGag / Pol, pRev, and pVSV-G, are formed, and there are also nucleic acid vectors that can place the target gene sequence. This system greatly reduces the possibility of unexpected production of active virus. Such vector systems may contain inducible genes, the most representative of which is the tetracycline-inducible system, which is the combination product of a regulatable system and a lentiviral vector, and artificially controls the expression of a target gene transplanted into a lentivirus, allowing both conditional gene expression and gene knockout, while still retaining the advantages of its own inactivation (Meng F et al., (2014) Advances of lentiviral vectors. Zhongguo Fei Ai Za Zhi 17(12):870-6; Martinez-Molina, E. et al., (2020) Large-Scale Production of Lentiviral Vectors: Current Perspectives and Challenges. Pharmaceutics 12:1051).

[0078] Other types of lentiviral vector systems mainly include simian immunodeficiency virus (SIV) vector system, feline immunodeficiency virus (FIV) vector system, equine infectious anemia virus (EIAV) vector system, caprine arthritis-encephalitis virus (CAEV) vector system, bovine immunodeficiency virus (BIV) vector system, ovine demyelinating virus (VMV) vector system, etc.

[0079] 2. Construction of Viral Vectors

[0080] 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.

[0081] 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.

[0082] The host cells required for viral vector production are also called producer / packaging cell lines. In the context of lentiviral vectors, the term "packaging cell line" refers to a cell line into which gag, pol and env genes have been introduced, and "producer cell line" refers to a cell line into which gag, pol, env genes and a foreign gene have been introduced. In one embodiment, a "packaging cell line" is a cell line into which gag, pol and env genes have been inserted into the cell genome, and a "producer cell line" is a cell line into which gag, pol, env genes and a foreign gene have been inserted into the cell genome. In another embodiment, a "packaging cell line" is a cell line into which gag, pol and env genes have been stably inserted into the cell genome, and a "producer cell line" is a cell line into which gag, pol, env genes and a foreign gene have been stably inserted into the cell genome.

[0083] Host cell culture can be divided into adherent culture and suspension culture. To increase the production of viral vectors, the total surface area to which the cells can attach can be increased, or the cells can be cultured in suspension.

[0084] HEK293 cells, also known as human embryonic kidney cells 293, are the most commonly used cells for the production of retroviruses and other viral vectors. HEK293T cells with SV40 T antigen integrated can produce higher viral titers than the parent cell line HEK293. HEK293T cells are usually cultured adherently in cell culture flasks containing 10% fetal bovine serum medium, resulting in low viral yields. To increase the surface area on which cells can attach, 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 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 iCELLis 500 is approximately 3000 HYPERFlasks.

[0085] Various equipment upgrades have made it possible to expand serum-dependent adherent HEK293T cells, but this has been time-consuming and labor-intensive. 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. Since then, new such cell lines, such as HEK293SF-3F6, have been developed (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 walls, being easier to subculture, and being easier to scale up.

[0086] For cell and gene therapy development companies, the demand for viral vectors during preclinical research, IND filing and clinical trials is limited, so lentivirus vectors are mostly produced by the method of transient transfection of adherent cells HEK293T. The adherent culture of HEK293T 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 large. One way to obtain a large growth area and reduce the amount of work is to adapt the cell line producing lentivirus to suspension culture. Compared with adherent culture, suspension culture can greatly improve the cell density.

[0087] In the second step, the viral vector expression system is introduced into a host cell, ie, the host cell is transfected with a packaging nucleic acid vector, an envelope nucleic acid vector, a helper nucleic acid vector, a nucleic acid vector carrying a heterologous gene, or the like.

[0088] In the present application, the term "viral expression system" 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 vector may further comprise a nucleic acid vector carrying a heterologous gene, whereby the nucleic acid encoded thereby, e.g., RNA or polypeptide / protein, can be packaged together into a viral vector particle. For example, a lentiviral expression system typically comprises a gag-pol gene, an env gene, and a helper gene rev, etc. In one embodiment, the lentiviral expression system comprises a nucleic acid vector comprising a HIV-1 gag-pol-rev gene, and a nucleic acid sequence encoding vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof. In another embodiment, the lentiviral expression system comprises a nucleic acid vector comprising a HIV-1 gag-pol gene, an HIV-1 rev gene, and a nucleic acid sequence encoding vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof. An AAV expression system typically comprises polynucleotides encoding AAV rep and cap, helper genes, and a rAAV genome. The term "nucleic acid vector" as used herein refers to a DNA or RNA vector, and in the embodiment of the present invention, may be a plasmid, or a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a P1-derived artificial chromosome (PAC), a fosmid, or a cosmid. The nucleic acid vector may contain an origin of replication, a promoter, a transcriptional regulatory element, or the like, as necessary. Any element may be operably linked to the promoter so that expression can be controlled. The promoter referred to herein may include a known promoter (complete or partial), which may be persistently activated or inducible, for example, in the presence of a regulatory protein. In one embodiment, the nucleic acid vector also includes an efficient promoter, such as a CMV promoter. The promoter has the advantage of promoting high-level expression of elements encoded on the non-mammalian nucleic acid vector. In another embodiment, the CMV promoter includes a sequence derived from the human cytomegalovirus strain AD169.The sequence can be obtained from Genome Accession No. X17403, e.g., base pairs 173731-174404. In the present application, a nucleic acid vector carrying a foreign gene may be referred to as a transfer vector, and in one embodiment, may be a transfer plasmid. A transfer vector plasmid typically includes a promoter (e.g., CMV), a 3' LTR (which may or may not be self-inactivated (i.e., SIN) 3'-LTR), a 5' LTR (which may or may not include a U5 region), an encapsidation sequence (ψ), and a potential foreign gene linked to the promoter.

[0089] "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.

[0090] 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.

[0091] 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.

[0092] 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 values. The PEI method can be used for cells that are cultured in adherence 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).

[0093] Lipofectamine-mediated transfection is the most efficient, the most convenient, suitable for various cell types, and has a very high transfection rate, e.g., in some cases, 100% of cells are found to be transfected with lentivirus. However, for large-scale viral vector production, the cost is high when using Lipofectamine.

[0094] 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.

[0095] It has been reported that the addition of sodium butanoate to the medium after transfection can increase the virus titer.

[0096] Of the above transfection methods, PEI is more suitable for adherent cultured cells, flow electroporation is more suitable for suspension cultured cells, and calcium phosphate and Lipofectamine are more suitable for small-scale applications due to cost reasons.

[0097] Once inside the host cell, the nucleic acid vector randomly integrates into the endogenous genome of the mammalian host cell. Therefore, it is necessary to select host cells that have integrated the nucleic acid encoded on the nucleic acid vector using an antibiotic resistance selection marker, e.g., a zeocin resistance marker.

[0098] 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 the nucleic acid vector is naturally circular. The nucleic acid vector may further comprise a region that shares homology with the 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, a method of inducing targeted cleavage of 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 an engineered cleavage system to repair the break 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 a prokaryotic 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 said at least one nuclease cleaves the host cell's genome and integrates the nucleic acid sequence into the cell's genome.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.

[0099] Transfection can also be divided into transient transfection and stable transfection. In the case of transient transfection, the foreign gene is expressed but not integrated into the cell genome and not replicated. The expression time of the foreign gene transiently expressed in the cell is limited, and usually lasts only a few days before the foreign gene is lost due to various factors during the cell division process. Stable transfection is based on transient transfection and requires only one important accidental process, that is, 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.

[0100] 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 expressed directly 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 genetic regulatory mechanism studies. Stable transfection is longer and more laborious than transient transfection.

[0101] Stable transfection cell lines are divided into constitutive stable transfection cell lines and inducible stable transfection cell lines. Constitutive stable transfection cell lines usually do not express highly cytotoxic viral envelopes, such as VSV-G of lentivirus, and produce viral vector particles at higher titers over several months. There are very few such cell lines, and lentivirus stable transfection cell lines include STAR cell lines, WinPac cell lines, RD-MolPack cells, and LentiPro26 cell lines. Inducible stable transfection cell lines usually induce the expression of the viral envelope only when it is necessary to produce virus, for example, when an inducer such as an antibiotic is added to the growth medium. Inducible stable transfection cell lines can also produce high titers of virus within a long period of time. For example, ProSavin can produce up to 3 × 10 5Such a cell line developed by Milani et al. can produce titers of 4.4 × 10 6 It can produce titers of TU / ml (Milani, M. et al., (2017) Genome Editing for Scalable Production of Alloantigen-free Lentiviral Vectors for in Vivo Gene Therapy. EMBO Mol. Med. 9:1558-1573).

[0102] Due to the difficulty in obtaining stably transfected cell lines, the current mainstream virus production, for example lentiviral vector production, uses the HEK293T cell line, which is also suitable for suspension culture.

[0103] The third step in viral vector production is to culture and amplify the host cells into which the viral vector expression system has been introduced, which is similar to the first step.

[0104] The fourth step is to isolate and purify the viral vector. The collected cell culture supernatant, which contains 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. 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 using 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.

[0105] Conventional large-scale viral vector purification methods include clarification, concentration and purification.

[0106] 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.

[0107] The clarified viral vector particles can be concentrated and permeated with a suitable buffer using tangential flow filtration (TFF), which involves using membranes with pore sizes of 1-100 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 with 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 90-100% of lentiviral particles can be recovered by TFF. Currently, TFF can process approximately 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).

[0108] For pharmaceutical products applied to humans, lentiviral vector products need to be further purified by chromatography. Anion exchange chromatography (AEX) is an efficient tool for purifying viral vectors, especially lentiviral vectors, based on the characteristic that lentiviral vectors are positively charged at neutral pH. When the viral vector supernatant passes through a column of a negatively charged matrix, the positively charged viral particles bind to the negatively charged matrix, and the impurities flow directly through the column. The viral vector is then exposed to a high salt environment (0.5-1 M 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).

[0109] 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 found. Heparin is an affinity ligand that is cheap and interacts with various virus types, and has been used in lentiviral vector purification, which can recover up to 53% of the viral vector and remove up to 94% of protein impurities and 56% of residual DNA. There is an interaction between the vector and heparin between the positively charged molecules on the surface of the virus particle and the negatively charged heparin, so NaCl is usually used to elute the vector from the heparin column. Usually, the viral vector cannot be eluted unless the salt concentration reaches about 0.5 M. Therefore, an additional washing or purification step, such as adding additional TFF, is required to remove the salt. Studies have shown that heparin does not interact with viral envelope proteins and can therefore bind to various virus types without causing yield loss.

[0110] 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.

[0111] After the purification step, the lentiviral vector needs to be frozen and stored at -80°C. According to reports, the half-life of lentiviral vectors is 1-2 days at room temperature and about 8 days at 4°C (Higashikawa, F. et al., (2001) Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors. Virology 280:124-131), and they can be stored at -80°C for 1-2 years (Valkama, AJ et al., (2020) Development of Large-Scale Downstream Processing for Lentiviral Vectors. Mol. Ther. Methods Clin. Dev. 17:717-730). Before cryopreservation, the cryopreservation solution must be replaced, and the sucrose and magnesium chloride components can reduce the loss of functional viral particles and improve storage stability (Valkama, AJ et al., (2020) supra; Bandeira, V. et al., (2012) Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks. Hum. Gene Ther. Methods 23:255-263; Kumru, OS et al., (2018) Physical Characterization and Stabilization of a Lentiviral Vector Against Adsorption and Freeze-Thaw. J. Pharm. Sci. 107: 2764-2774).

[0112] In this application, specifically, PowerS TM A method for producing viral vector particles using the cell line of the present application, including 293T, i) culturing the cell line; ii) introducing a viral vector expression system into the cell line; iii) culturing the cell line under conditions in which viral vector particles are produced; and vi) harvesting the viral vector particles.

[0113] 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., 8% CO. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0114] Step i) may further comprise passaging the cell line.

[0115] 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.

[0116] The viral vector expression system may include a packaging nucleic acid vector and an envelope nucleic acid vector. The viral vector expression system may further include a helper nucleic acid vector. The viral vector expression system may further include a nucleic acid vector comprising a heterologous nucleic acid, for example a transfer nucleic acid vector comprising a heterologous nucleic acid. These nucleic acid vectors may be plasmids.

[0117] The length of the heterologous nucleic acid is 4 kb or less. The heterologous nucleic acid can encode a protein such as a polypeptide or a chimeric antigen receptor, or a nucleic acid such as an siRNA. In one embodiment, the heterologous nucleic acid can encode a chimeric antigen receptor (CAR), for example, a CAR that targets CD19, CD20, CD22, CD30, CD33, or CD269 (BCMA). In one embodiment, the CAR encoded by the heterologous nucleic acid can target an antigen involved in growth and differentiation signaling, including carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR, HER2), tyrosine kinase receptor (EPHA2), and the like. In one embodiment, the CAR encoded by the heterologous nucleic acid can target an antigen involved in tumor vascular development, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and the like. In one embodiment, the CAR encoded by the heterologous nucleic acid can target tumor stroma and extracellular matrix antigens of tumor supporting structures, including fibroblast activation protein (FAP), tenascin protein, and the like. Plasmids containing heterologous nucleic acid may include pCDH and pCMV, among others.

[0118] The viral vector may be a retroviral vector, an adenoviral vector, or an adeno-associated viral vector. The retroviral vector may be a lentiviral vector, an alpha-retroviral vector, a gamma-retroviral vector, or a foamy retroviral vector. In one embodiment, the retroviral vector is a lentiviral vector selected from HIV-1, HIV-2, SIV, FIN, EIAV, and VISNA. In one embodiment, the lentiviral vector may be an HIV-1 vector. In another embodiment, the viral vector may be an adeno-associated viral vector, such as AAV2, AAV5, AAV8, AAV9, and AAV10.

[0119] The retroviral vector expression system may include a vector containing a retroviral structural protein gag-pol gene and a nucleic acid vector containing the env gene or a functional substitute thereof. The env gene or a functional substitute thereof may encode the vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof. The retroviral vector expression system may further include a nucleic acid vector containing the helper gene rev or a gene similar thereto. The retroviral vector expression system may further include a nucleic acid vector containing a heterologous nucleic acid, for example a transfer nucleic acid vector containing a heterologous nucleic acid.

[0120] In one embodiment, a method is provided for producing a lentiviral vector, comprising introducing into a cell line three nucleic acid vectors, each comprising the HIV-1 gag-pol-rev genes, a nucleic acid encoding vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof, and a heterologous nucleic acid.

[0121] In one embodiment, a method for producing a lentiviral vector includes introducing into a cell line four nucleic acid vectors, each of which includes an HIV-1 gag-pol gene, an HIV-1 rev gene, a nucleic acid encoding vesicular stomatitis virus glycoprotein (VSV-G) or a derived protein thereof, and a heterologous nucleic acid. The nucleic acid vector including the heterologous nucleic acid may include a self-inactivating lentiviral long terminal repeat.

[0122] Step iii) may include suspension culture of the cell line. The cell line may be cultured under any suitable conditions for the cell line to produce the viral vector, as 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, 8%. The serum-free medium may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0123] Step iii) may further comprise passaging the cells.

[0124] This application relates to PowerS, a viral vector expression system. TM - Bench and pilot batch production is performed on 293T host cells, cultured, for example, in 300 ml shaker flasks, and in suspension culture in, for example, 1 L, 2 L, 10 L, 50 L and 200 L bioreactors (e.g., WAVE reactors or glass reactors) to produce viral vectors.

[0125] The method may further comprise performing titer detection on the viral vector after step vi).

[0126] Viral vector applications

[0127] The viral vector expression system may comprise a nucleic acid vector comprising a heterologous nucleic acid, e.g., a transfer vector plasmid comprising a heterologous nucleic acid, for encoding a therapeutic or research protein, such as a chimeric antigen receptor, or a therapeutic or research nucleic acid, such as a siRNA. The application of the viral vector depends mainly on the protein and nucleic acid that the heterologous nucleic acid encodes.

[0128] 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.

[0129] 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-alpha), platelet-derived growth factor (PGF-alpha), and the like. These include, but are not limited to, any member of the transforming growth factor beta superfamily, including growth factors (PDGF), insulin-like growth factors 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.

[0130] 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.

[0131] Other useful heterologous nucleic acid products include gene products capable of correcting inborn errors of metabolism, such as carbamoyl synthetase I, carbamoyl synthetase, carbamoyl synthetase, carbamoyl synthetase, carbamoyl synthetase, α-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, clotting factors, such as factor V, factor VIIa, factor VIII, factor IX, factor X, factor XIII or protein C, cystathionine-β-cysteine, Examples of such cDNA sequences include, but are not limited to, isovaleryl A dehydrogenase, 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, hepatic phosphorylase, glycine decarboxylase, H protein, T protein, glycine decarboxylase (CFTR) sequence, and dystrophin cDNA sequence.

[0132] 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. syltransferase, 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 factor 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 ... and the like), suicide gene products (e.g., herpes simplex virus thymidine kinase, cytosine deamidase, diphtheria toxin, cytochrome P450, deoxycytidine cytokinase, tumor necrosis factor, and the like), 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)), immunomodulatory peptides, resistance or immunogenic peptides, or The 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.

[0133] The heterologous nucleic acid can further encode a tumor associated antigen (TAA). Non-limiting examples of TAAs include testicular tumor associated specific antigens (e.g., MAGE, BAGE, and GAGE), melanocyte differentiation antigens (e.g., tyrosinase, Melan-A / MART-1), CDK4, MUM-1, β-catenin, gp100 / pmel17, TRP-1, TRP-2, MITF, MITF-A, and MITF-M. Other non-limiting examples of TAAs include melanoma GP75, annexin I, annexin II, adenosine deaminase binding protein ((ADAbp), PGP9.5 (Rode et al., 1985). Histopathology 9:147), colorectal-related antigen (CRC)-C017-1A / GA733, Ab2BR3E4, CI17-1A / GA733, Hsp70, Hsp90, Hsp96, Hsp105, Hsp1 10, HSPPC-96, stress protein gp96, gp96-related cellular peptide, G250, dipeptidyl peptidase IV (DPPIV), thyroid globulin, STn, carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA) peptide CAP-1, carcinoembryonic antigen (CEA) peptide CAP-2, etv6, aml1, prostate-specific antigen (PSA), PSA antigenic determinants PSA-1, PSA-2, PSA-3, Ad5-PSA, parathyroid hormone-related protein (PTH-rP), EGFR, PLU1, carcinoembryonic antigen-immature laminin receptor (OFA-iLR), MN / CAIX (CA9), HP59, cytochrome oxidase 1, sp100, msa, RanGTPase activating protein, Rab-GAP protein, PARIS-1, T cell receptor / CD3-ζ chain, cTAGE-1, SCP-1, glycolipid antigen-GM2, GD2 or GD3, GM3, FucosylGM1, glycoprotein antigen-Tn, Sialyl-Tn, TF and Mucin-1, CA125 (MUC-16), MAGE family antigen, GAGE-1,2, BAGE, RAGE, LAGE-1, GnT-V, MUM-1, EP-CAM / KSA, CDK4, MUC family antigen, HER2 / neu, ErbB-2 / neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, NeuGcGM3, Fos-related antigen, cyclophilin B, RCAS1, S2, L10a, L10a, telomerase rt peptide, cdc27, p120ctn, PRAME, GA733 / EoCam, NY-BR-1, NY-BR-2, NY-BR-3, NY-BR-4, NY-BR-5, NY-BR-6, NY-BR-7, NY-ESO-1, L19H1, MAZ, PINCH, PRAME, Prp1p / Zer 1p, WT1, adenomatous polyposis coli (APC), PHF3, LAGE-1, SART3, SCP-1, SSX-1, SSX-2, SSX-4, TAG-72, TRAG-3, MBTAA, Smad tumor antigen, lmp-1, HPV-16E7, c-erbB-2, EBV-encoded nuclear antigen (EBNA)-1, herpes simplex virus thymidine kinase (HSVtk), alternative splice isomer of XAGE-1, TGFbetaRII frameshift mutation, BAX frameshift mutation.

[0134] The heterologous nucleic acid can also encode a gene product, such as CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, CEA, EGF-RIII (epidermal growth factor receptor variant 3), EGP-2, erb-B2, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, GD2, Her2 / neu, IL-13R-a2, KDR, k light chain, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, NKG2D, carcinoembryonic antigen (h5T4), PSCA, prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate epithelial cell-derived Ets transcription factor (PDEF), TAA targeting mAbIgE, TAG-72, and VEGF-R2.

[0135] Alternatively, the heterologous nucleic acid may include, for example, siRNA, antisense molecules, and miRNA. Clinically useful lentiviral vectors can also express antisense genes for human immunodeficiency virus (HIV) (Levine BL et al., (2006) Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci US A. 103(46):17372-7) and other important human pathogens. Naldini (Naldini L. (2011) Ex vivo gene transfer and correction for cell-based therapies. Nat. Rev. Genet. 12:301-315) has been cited for discussing these and other useful applications of lentiviruses and related vectors. Antisense genes contained in lentiviral 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 and 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 (types 1, 2, 3, 6, 7, 8, 12, 17) in spinocerebellar ataxia, FMR1 (Fragile X Syndrome) in 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 Mechanism of Development 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, HIVTAR, human immunodeficiency virus transactivator response element gene, C-C 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 tumors, apoptosis inhibitor B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia, nucleotide reductase M2 (RRM2) in solid tumors, Furin in solid tumors, Polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol transferase 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.

[0136] In one embodiment, the heterologous nucleic acid can encode a chimeric antigen receptor (CAR), for example, a CAR that targets CD19, CD20, CD22, CD30, CD33, or CD269 (BCMA). In one embodiment, the CAR encoded by the heterologous nucleic acid can target an antigen involved in growth and differentiation signaling, including carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR, HER2), tyrosine kinase receptor (EPHA2), and the like. In one embodiment, the CAR encoded by the heterologous nucleic acid can target an antigen involved in tumor vascular development, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and the like. In one embodiment, the CAR encoded by the heterologous nucleic acid can target tumor stroma and extracellular matrix antigens of tumor supporting structures, including fibroblast activation protein (FAP), tenascin protein, and the like. Plasmids containing heterologous nucleic acids may include pCDH and pCMV, and the like.

[0137] Host cell line screening

[0138] 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.

[0139] Serum-free culture

[0140] In general, the growth of all animal cells depends on the presence of serum. Most cells cannot grow without the addition of serum. Serum-free media is usually supplemented with bovine serum.

[0141] 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.

[0142] 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.

[0143] 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:

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] Serum-free medium is formed by adding hormones, growth factors, attachment factors and binding proteins to a complete basal nutrient medium.

[0150] 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. MEM, DMEM, RPMI 1640, F12, and TC100 are the most widely used of the many synthetic media. Because the components of the basal media are completely known, some components of the basal media can be appropriately adjusted when culturing different cell lines to better match the nutritional requirements of the cell lines or improve the expression of target proteins.

[0151] 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.

[0152] 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 vitro. 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 through 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.

[0153] 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.

[0154] 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, such as fibronectin, collagen, laminin, polylysine, etc. In suspension culture such as that of the present application, there is no need to add adhesion factors to the medium.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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 , LifeTechnologies), LV-MAX (A35834-01, Gibco TM ), Expi293 (Gibco TM , A14351-01), EX-CELL (registered trademark) 293 (Sigma, 14571C-1000mL), Banlan CD HEK293 (Irvine, 91165), Pro293s-CDM (Lonza, 12-765Q), OPM-293 CD03 (Okuura Susumu, 81070-001), and Celkey ​​CD HEK293 (Kenjun, 10804-19008) medium.

[0159] Suspension culture

[0160] Suspension culture refers to the culture of single cells and small cell pellets by dispersing and suspending cells in a medium, which 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.

[0161] According to cell adhesion, suspension culture technology can be divided into suspension cell culture process and adherent cell microcarrier suspension culture process. Suspension cells (such as CHO cells, BHK21l 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, and the contamination rate and cost are low. On the other hand, when adherent cells are cultured in a reactor, they need to use microcarriers, 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. Although the process is complicated compared to suspension culture, microcarrier culture has great advantages over rotary flask culture, and it can improve the production scale, product quality and work efficiency, so it is the main means of suspension culture of adherent cells.

[0162] 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 recovered 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 viral 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.

[0163] 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 appropriate heating or cooling methods must be used to control the culture temperature at 35°C to 37°C 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 adversely affect cell growth.

[0164] 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.

[0165] In the liquid suspension culture process, it is important to subculture the cells in a timely manner, as the culture will enter a stationary phase of division after a period of growth. For most suspension cultures, the cells will reach maximum density on the 18th to 25th day of culture, at which point they should be subcultured for the first time. During subculture, large cell clumps and inoculum residues should be removed.

[0166] Cell line screening

[0167] The essence of cell screening acclimation is to apply modern cell biology technology and study aspects such as adaptability to different culture methods (e.g., suspension), different media (e.g., serum-free media), reduction of apoptosis rate, improvement of cell vitality, 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, cell vitality during acclimation must be maintained at 90% or more. The proliferation ability of cells directly affects 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, vitality, 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.

[0168] In suspension culture, cells are prone to clumping. When cells clump, many of the plasmids used to produce vectors cannot be transfected into the cells, and the cells between the large clumps may die due to lack of nutrients. Currently, the method of improving cell clumping is mainly by adding anti-clumping agents. The addition of anti-clumping agents significantly reduces the transfection efficiency, resulting in the cells being unable to be transfected with packaging lentivirus.

[0169] 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.

[0170] In the present application, the inventors have primarily improved the adaptation / screening methods for HEK293T cells, which are currently most commonly used for lentivirus production.

[0171] The inventors believe that the use of a suspension culture method in which adherent cells cultured in normal medium are directly added to serum-free medium to perform suspension culture, without using the industry-standard culture step of gradually reducing the serum concentration from high-concentration serum culture, significantly saves time, and has high dispersibility (i.e., less agglomeration), high growth density, and may also contribute to some extent to the success of screening for cell lines that produce high levels of viral vectors.

[0172] In addition, the inventors of the present application have further found that the use of some 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 with high dispersibility, i.e., non-aggregation. Specifically, the cells screened using these three media can reach a high density (~1E7 cells / ml) and have a low proportion of aggregated cells, which is advantageous for screening non-aggregated subclones.

[0173] The present application mainly screened HEK293T cells, which are commonly used for producing lentiviral vectors, because they have rapid proliferation, high transfection efficiency, can efficiently produce viral vectors, and have the potential to produce viral vectors on a large scale.

[0174] Specifically, the present application provides a method for screening or adapting a HEK293T cell line suitable for serum-free suspension culture, the method comprising: i) taking HEK293T cells cultured in an adherent culture medium containing serum, and placing them in a serum-free medium to culture them in suspension; and ii) selecting a monoclonal cell line suitable for serum-free suspension culture.

[0175] The serum-containing medium in step i) may contain 10% serum, for example 10% fetal bovine serum.

[0176] The suspension culture in step i) may comprise a shaking culture at 37° C. and 8% CO 2 .

[0177] The suspension culture in step i) may include passaging of the cells.

[0178] The serum-free medium may be selected from LV-MAX Production Medium (Gibco, A35834-01), Expi293 Expression Medium (Gibco, A14351-01), FreeStyle™ 293 Expression Medium (Gibco, 12338-018), CD293 AGT Medium (Gibco, 11913-019), EX-CELL® 293 Serum-Free Medium (Sigma, 14571C-1000mL), Banlan CD HEK293 Medium (Irvine, 91165), Pro293s-CDM Serum-Free Medium (Lonza, 12-765Q), OPM-293 CD03 Medium (Okuura Susumu, 81070-001) and Celkey ​​CD HEK293 Medium (Kenjun, 10804-19008), preferably LV-MAX Production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0179] Step ii) may involve selecting monoclonal cell lines that are suitable for serum-free suspension culture and are highly dispersible (i.e., do not aggregate).

[0180] Step ii) may comprise culturing cells in a semi-solid medium, selecting single cells from the semi-solid medium, and culturing the selected cells in suspension.

[0181] 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.

[0182] The semi-solid medium may be a methylcellulose-based medium supplemented with screening medium.

[0183] In the process of carrying out the method, there is no need to add any anti-agglomerating agent.

[0184] In the method of the present application, 1) cells cultured in an adherent culture medium in serum-containing medium are directly cultured in serum-free medium, and there is no need to gradually reduce the amount of serum, thereby shortening the screening process; 2) cells are cultured in 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 is used, which is more advantageous for screening HEK293T cell lines that are suitable for suspension culture, i.e., have 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) the use of semi-solid medium facilitates the screening of monoclonals that meet the conditions.

[0185] semi-solid culture

[0186] In addition, the inventors of the present application performed monoclonal cell screening using a semi-solid culture method, rather than the finite dilution method, in the above screening method.

[0187] Finite dilution is a cell cloning method commonly used in laboratories. However, the finite dilution method may add multiple cells in one well, so subcloning must be performed multiple times to improve the probability of monoclonality. In addition, when screening subclones by finite dilution in an experiment, a single cell in a well plate cannot grow normally, which may be related to the characteristics of the cell itself. In contrast, semi-solid medium is a faster and easier cloning method, in which a single cell is fixed in a semi-solid medium and grows into a discrete monoclonal cell group, which is advantageous for tracking cell growth. And the monoclonal cells can be transferred to a well plate after being amplified to a certain number, which improves the problem that a single cell cannot grow normally. In addition, the semi-solid medium can be used to inoculate multiple cells in the same well, which can reduce the amount of work for the operator, and compared with the finite dilution method, the number of culture well plates can be reduced, and reagents, consumables and culture box space can be saved.

[0188] Semi-solid medium is often used for culturing hybridoma and CHO cells, and allows cell cloning to be performed more efficiently. The application of semi-solid culture in screening viral vector packaging / producing cells has not yet been reported.

[0189] Host Cell Lines of the Present Application

[0190] From HEK293T 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.

[0191] One of them is PowerS TM It was named .-293T and deposited at the China General Microorganism Collection Center (CGMCC) under the Budapest Treaty on December 7, 2020, with the deposit number CGMCC No.: 21110.

[0192] PowerS TMThe OPM-293T cell line can be grown in suspension culture in serum-free medium, which may be selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium.

[0193] PowerS TM -293T cell line may not have any cell clumps during the suspension culture process.

[0194] PowerS TM -293T cell line can be used for production of viral vectors.

[0195] PowerS TM The 293T cell line can grow in suspension culture in serum-free medium, with high proliferation density, good vitality, no cell clumps, i.e., high dispersibility, and therefore no need to add additional anti-aggregation agents during the suspension culture process.

[0196] PowerS TM In OPM-293 CD03 medium, the doubling time of 22 hours was short, and the cell density in log phase was 5–7 × 10 6 cells / ml, with a maximum density of 1.48 × 10 7 cells / ml.

[0197] Compared to Gibco's CTS trademarked virus-producing cells (publication number: MAN0018590), PowerS TM -293T has a short cell doubling time and produces more viral vector particles after transfection with viral plasmids.

[0198] 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.

[0199] Example 1. Serum-free suspension adaptation of HEK293T cells

[0200] HEK293T cells purchased from ECACC were incubated in a 37°C water bath for less than 1 minute to rapidly thaw the cells in the cryopreservation tube until there were no ice chunks in the cryopreservation tube.

[0201] The cells were transferred from the cryopreservation tube to a sterile centrifuge tube, 4-5 ml of prewarmed DMEM (containing 10% FBS) was added, and the tube was centrifuged at 200 g for 5 minutes.

[0202] The supernatant in the culture flask was discarded, and the cell pellet was resuspended in 10 ml of DMEM (containing 10% FBS). The resuspended cells were added to a T75 culture flask, and the T75 culture flask was placed in a culture box at 37° C. and 5% CO2.

[0203] The cells were incubated for three days, and when the cell confluence was >90%, the cell medium in the culture flask was discarded. Here, the cell confluence refers to the percentage of the surface area of ​​the culture vessel covered by the monolayer cells observed under a microscope, relative to the monolayer grown cells. The adhesion layer was washed once with 5 ml of DPBS. The cells were incubated with 0.25% trypsin (Gibco, 12563-029) for 2 minutes, and after the cells were detached, 4-5 ml of 10% FBS-containing DMEM was added to the T75 culture flask and cultured.

[0204] The cells were transferred to a centrifugal tube and centrifuged at 200 g for 5 min.

[0205] The supernatant was aspirated, the cell pellet was resuspended in 10 ml of DMEM (containing 10% FBS), the resuspended cells were added to a T75 culture flask, and the T75 culture flask was placed in a culture box at 37° C. and 5% CO 2 .

[0206] Cells were passaged when they were fully adapted to adherent growth and reached 90% confluency.

[0207] The cells were digested with 0.25% trypsin and centrifuged at 200 g for 5 min. The supernatant was aspirated and fresh serum-free medium (SFM) was added to the cell pellet to resuspend the cells. There are nine types of serum-free media in total, including LV-MAX Production Medium (Gibco, A35834-01), Expi293 Expression Medium (Gibco, A14351-01), FreeStyle™ 293 Expression Medium (Gibco, 12338-018), CD293 AGT Medium (Gibco, 11913-019), EX-CELL® 293 Serum-Free Medium (Sigma, 14571C-1000mL), Banlan CD HEK293 Medium (Irvine, 91165), Pro293s-CDM Serum-Free Medium (Lonza, 12-765Q), OPM-293 CD03 Medium (Okuura Susumu, 81070-001), and Celkey ​​CD HEK293 Medium (Kenjun, 10804-19008). The cells in serum-free medium were transferred to a 125 ml shaker flask and placed in a culture box at 37° C. and 8% CO 2 , and cultured in a shaker with a shaking diameter of 19 mm and a rotation speed of 125 rpm.

[0208] Cell density is 3-6×10 6 When the cells reached a density of 0.35-0.55×10 cells / ml, they were subcultured. 6 cells / ml and incubated for 3-4 days until viability exceeded 95%, where viability was determined by a VI-CELL cell counting instrument (BECKMAN).

[0209] The cells cultured in the nine types of serum-free media were placed in cell culture plates, and the colony morphology was observed by photographing them under a microscope, as shown in Figure 1.

[0210] Here, the cells acclimatized in LV-MAX production medium (Gibco, A35834-01), Expi293 expression medium (Gibco, A14351-01), and OPM-293 CD03 medium (Susumu Okuura, 81070-001) had higher density and better dispersibility. The cells acclimatized in these three media were taken and growth curves were measured. Specifically, 0.4±0.5×10 6The cells were inoculated into a cell culture flask at a cell density of 1×10 cells / ml, marked as D0, and 600 microliters of cell suspension were aspirated for cell counting. Then, the cells were cultured in suspension for 8 days, and 600 microliters of cell suspension were aspirated for cell counting every day. The cell growth curves were plotted and shown in Figure 2. As shown in Figure 2, the cell growth density and viability trends in the above three media were consistent, and the maximum cell density was 1×10 7 cells / ml and cell viability was greater than 90%.

[0211] Cells that could reach higher densities were selected by subculture, and ultimately reached a suspension density of up to 1.2×10 in the three media. 7 Cell lines were selected with a final density of 1×10 cells / ml. 7 The cells were cryopreserved in a serum-free suspension culture of screened cell lines at viable cells / ml, and may contain 10-20 cell aggregates.

[0212] Example 2. Performance optimization of serum-free suspension-adapted HEK293T

[0213] To further solve the problem of aggregation of serum-free suspension-adapted HEK293T cells, cells adapted to LV-MAX production medium, Expi293 and OPM-293 CD03 medium were cloned using semi-solid matrix adhesive. The specific steps are as follows:

[0214] The serum-free suspension acclimatized cells obtained in Example 1 were diluted correspondingly using the above three serum-free media to obtain a density of 5×10 3 A cell suspension of 100 cells / mL was made.

[0215] 100, 200, 300, or 400 μL of each cell suspension was added to each of the 2.0 mL cryopreservation tubes containing 1 mL of semi-solid matrix adhesive (Stemcell, 03816) to form a cell concentration gradient, i.e., 4 cell gradients were formed for each cell, and the total cell amount in each well was 500, 1000, 1500, and 2000 cells, respectively. The tubes were covered and shaken for 1-2 min. The medium was opaque. The medium was left to stand for 2-5 min, allowing the medium to collect at the bottom of the tube and for air bubbles to rise to the liquid surface.

[0216] The cells were inoculated using a 5 mL disposable syringe. Specifically, 1 to 1.5 mL of the above semi-solid cell suspension was added to each culture well of a 6-well plate, and each density was repeated three times. The culture plate was gently tilted and rotated to ensure that the medium was evenly distributed at the bottom of the culture well.

[0217] The covered 6-well plates were placed into a similarly covered larger culture dish, which also contained a 100 mm culture dish without a lid containing sterile water. Alternatively, half the height of sterile water or PBS was added into the groove between each well of the culture plate before covering the 6-well plate. The colony status in the culture was checked based on the doubling time of the cell line. The colonies were visible to the naked eye and were generally 0.5-1.0 mm in diameter. The number of colonies grown under each seeding density was counted. The culture plates were photographed to preserve the colony morphology.

[0218] After 7 days, 24-well plates were prepared and 1 mL of the corresponding serum-free medium was added to each well plate. 5 μL of the corresponding cell-adapted serum-free medium was aspirated using a 10 μL gun head, and the large clones in the 6-well plate culture were lightly aspirated, and the cells were blown onto the 24-well plate and observed under a microscope to see whether they were single cells. The 24-well plate was placed in a culture box at 37°C and 8% CO2, and cultured on a shaker with a rotation speed of 125 rpm and a shaking diameter of 19 mm.

[0219] The cells were replenished once every 3 days, and based on the cell amount, the cells were expanded into 6-well plates with 2 mL of medium per well, placed in a culture box at 37°C and 8% CO2, and cultured in a shaker with a shaking diameter of 19 mm and a rotation speed of 125 rpm.

[0220] Based on the cell quantity, the cells were amplified into a 125 mL shaker flask and subcultured three times, the cell suspension was taken and observed under a microscope, and the cell morphology was as shown in Figure 3. As can be seen from Figure 3, the non-aggregating cell line can be screened by clone. The non-aggregating cells were cryopreserved.

[0221] The cells conditioned from OPM-293 CD03 were analyzed using PowerS TM The cell line was named β-293T, and its cell morphology is shown in Figure 4A. The 8-day growth curve and growth activity diagram are shown in Figure 4B. The cell line did not aggregate, had a short cell doubling time of about 22 hours, and had a logarithmic cell density of 5-7 × 10 6 cells / ml with a maximum density of 1.48×10 7 cells / ml could be achieved.

[0222] In contrast, Gibco's cells, CTS trademarked virus producer cells (Publication Number: MAN0018590), had a doubling time of approximately 26 hours.

[0223] Example 3. Production of viral vectors using serum-free suspension-adapted HEK293T cells PowerS™-293T The PowerS™-293T cells obtained in Example 2 were cultured at 0.55 × 10 6 The cells / ml were inoculated into a 125 ml shaker flask and cultured in suspension for three days in a shaker with a shaking diameter of 19 mm and a rotation speed of 125 rpm in a culture box at 37°C and 8% humidity.

[0224] Take the cell suspension from the suspension cell culture shaker flask, count and confirm that the cell density is 5.5 x 10 6When the cell viability reached >95% and the number of cells / ml was reached, transfection was performed.

[0225] Taking a 30 ml transfection system as an example, first adjust the transfection cell density and then calculate the cell culture medium (transfection volume (30 ml) × transfection cell density (5 × 10 6 The cell culture flask was placed in a culture box at 37°C and 8% humidity, and cultured in suspension in a shaker with a shaking diameter of 19 mm and a rotation speed of 125 rpm. Before transfection, the cell density was adjusted to 5.5×10 6 The cells / ml were adjusted and placed in a culture box.

[0226] At the same time, a transfection complex was prepared.

[0227] Specifically, OPM-293 CD03 medium and PEIpro transfection reagent were gently shaken 4-5 times before use. 56.25 micrograms of plasmid in TE buffer was placed into 1.5 ml of OPM-293 CD03-containing medium preheated at 37°C, and the transfer plasmids: pMDLg.pRREKan (gag-pol, 8847 bp): pRSV-Rev-Amp (rev, 5779 bp): pMD2.gKan (vsvg, 4137 bp) = 4:2:2:1 were gently mixed 4-5 times, and labeled as tube 1-DNA. Here, the transfer plasmids were a total of three types, which were heterologous gene-containing pCDH vectors with sizes of 7545 bp, 9517 bp, and 9505 bp, respectively. 112.5 ml of PEIpro transfection reagent (Polyplus Transfection, PT-115-100) was added to 1.5 ml of OPM-293 CD03 medium preheated to 37°C, gently mixed 4-5 times, and labeled as tube 2-PEIpro. The transfection buffer in tube 2 was transferred to tube 1, repeatedly mixed with a gun 8-10 times or more, vortexed in a vortex meter as soon as possible for 10 seconds, and left to stand at room temperature for 15 minutes.

[0228] The resulting transfection complex was slowly added to the cell solution to be transfected, and the shaker flask was gently shaken while adding. After the operation was completed, the inoculated cell culture flask was placed in a culture box for suspension culture. After 24 hours of transfection, sodium butanoate was added to a final concentration of 5 mM. After 48 hours, the virus was harvested and the virus titer was detected using FACS.

[0229] Gibco's CTS™ virus-producing cells (Pub. No. MAN0018590) were used as a positive control, and all other procedures were identical to those of PowerS™-293T, except for the transfection reagent. Specifically, Gibco transfection was performed using the LV-MAX transfection kit (ThermoFisher, A35348).

[0230] The results of the transfection test are shown in Table 1. The virus yields of PowerS™ 293T cells were all higher than those of Gibco cells, and the virus titers after the test were all 1×10 7 was greater than TU / mL.

[0231] [Table 1]

[0232] The embodiments of the present invention are not limited to the description of the above examples, and those skilled in the art may make various modifications and improvements to the form and details of the present invention without departing from the spirit and scope of the present invention, all of which are deemed to be within the protection scope of the present invention.

Claims

1. Novel human embryonic kidney HEK293T cells deposited at the China Center for the Collection and Management of Ordinary Microorganisms, with accession number CGMCC NO.: 21100.

2. A method for screening HEK293T cell lines suitable for serum-free suspension culture, comprising: Step i) of removing HEK293T cells cultured in serum-containing medium as adherent cells and placing them in serum-free medium for suspension culture; ii) selecting a non-aggregating monoclonal cell line suitable for serum-free suspension culture; wherein step ii) comprises culturing cells in a semi-solid medium, selecting single cells from the semi-solid medium, and culturing the selected cells in suspension.

3. 3. The method of claim 2, wherein the serum-containing medium in step i) comprises 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. 3. The method of claim 2, wherein the suspension culture in step i) comprises passaging the cells.

6. The suspension culture of the selected cells in step ii) is maintained at 37°C, 8% CO ２ The method of claim 1, comprising shaking culture under the conditions:

7. 2. The method of claim 1, wherein the suspension culture of the selected cells in step ii) comprises passaging the cells.

8. 2. The method of claim 1, wherein the semi-solid medium is a methylcellulose-based medium supplemented with a screening medium.

9. 3. The method of claim 2, wherein the serum-free medium is selected from LV-MAX production medium, Expi293 expression medium, and OPM-293 CD03 medium.

10. The method according to any one of claims 2 to 9, wherein the method is carried out without the addition of an anti-agglomerating agent.

11. A cell line screened by the method according to any one of claims 2 to 10, wherein the cell line does not aggregate in a liquid medium.

12. Use of a medium selected from LV-MAX production medium, Expi293 expression medium and OPM-293 CD03 medium in screening for a non-aggregating HEK293T cell line suitable for suspension culture.

13. A method for producing a viral vector using the cell line of any one of claims 1 and 11, comprising: i) culturing said cell line in suspension; ii) introducing the viral vector expression system into the cell line; iii) culturing the cell line under conditions under which the viral vector was produced; vi) harvesting the viral vector.

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

15. The method of claim 13 , wherein the viral vector expression system comprises a packaging nucleic acid vector and an envelope nucleic acid vector.

16. The method of claim 13 , wherein the viral vector expression system further comprises a helper nucleic acid vector and / or a nucleic acid vector comprising a heterologous nucleic acid.

17. 17. The method of claim 16, wherein the heterologous nucleic acid is 4 kb or less in length.

18. 14. The method of claim 13, wherein the viral vector is selected from a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

19. 19. The method of claim 18, wherein the retroviral vector is a lentiviral vector.

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