WO2021218000A1 - 用于逆转录病毒载体的生产细胞和包装细胞及其制备方法 - Google Patents

用于逆转录病毒载体的生产细胞和包装细胞及其制备方法 Download PDF

Info

Publication number
WO2021218000A1
WO2021218000A1 PCT/CN2020/115522 CN2020115522W WO2021218000A1 WO 2021218000 A1 WO2021218000 A1 WO 2021218000A1 CN 2020115522 W CN2020115522 W CN 2020115522W WO 2021218000 A1 WO2021218000 A1 WO 2021218000A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
tre
gag
pol
transcription
Prior art date
Application number
PCT/CN2020/115522
Other languages
English (en)
French (fr)
Inventor
薛博夫
杨银辉
刘科
Original Assignee
深圳市深研生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市深研生物科技有限公司 filed Critical 深圳市深研生物科技有限公司
Priority to JP2022564610A priority Critical patent/JP2023523043A/ja
Priority to US17/766,684 priority patent/US20230051793A1/en
Priority to EP20934080.1A priority patent/EP4144856A1/en
Publication of WO2021218000A1 publication Critical patent/WO2021218000A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material
    • C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material
    • C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)

Definitions

  • the present disclosure relates to the field of viral vectors, specifically, to the field of retroviruses, especially lentiviral vectors; more specifically, to methods for preparing retroviruses, especially lentiviral vectors, for the preparation of retroviruses, especially lentiviral vectors. Production/packaging cells and methods of making the cells.
  • Retroviruses also known as retroviruses, belong to a class of RNA viruses. They are double-stranded RNA viruses with an envelope. The main feature is that they can "reverse transcribe" their genome from RNA to DNA.
  • the diameter of the virion is generally about 100 nm, and it contains a dimeric genome (two identical single-stranded positive-stranded RNAs) that forms a complex with the nucleocapsid (NC) protein.
  • NC nucleocapsid
  • Its genome is encapsulated in a protein capsid (CA), which also contains proteins with enzymatic activity, namely reverse transcription protease (RT), integrase (IN) and protease (PR), these enzymes are viral infections Necessary.
  • CA protein capsid
  • RT reverse transcription protease
  • PR protease
  • the matrix protein (MA) forms a layer outside the core of the capsid, which interacts with the envelope, which is a lipid bilayer derived from the host cell membrane and surrounding the core particle of the virus.
  • the envelope which is a lipid bilayer derived from the host cell membrane and surrounding the core particle of the virus.
  • Anchored on the envelope is the viral envelope glycoprotein (Env), which is responsible for recognizing specific receptors on the host cell and initiating the infection process.
  • the envelope protein is formed by two subunits, which are the transmembrane (TM) subunit that anchors the protein in the lipid membrane and the surface (SU) subunit that binds to cell receptors.
  • ⁇ -retroviral vector is the most commonly used retroviral vector, accounting for 17.3% of all transfection methods used in clinical trials of gene therapy applications in 2017.
  • people's interest in Lentiviral vector derived from complex retroviruses such as human immunodeficiency virus (HIV-1) is increasing day by day, from 2.9% of gene therapy clinical trials in 2012 to 7.3 in 2017 %.
  • Lentiviral vector derived from complex retroviruses such as human immunodeficiency virus (HIV-1)
  • HIV-1 human immunodeficiency virus
  • lentivirus can transduce non-dividing cells, which distinguishes it from other viral vectors (including gamma-retroviral vectors).
  • lentiviruses have a more favorable gene insertion site lineage compared to retroviruses.
  • retrovirus and lentiviral vector are: as a gene delivery tool, its gene delivery capacity can reach 9kb; smaller patient immune response and better clinically proven safety; high in vivo and in vitro Transduction efficiency; and the ability to permanently integrate foreign genes into the target cell genome, so that the delivered genes have long-term expression.
  • the prototype lentiviral vector system was developed based on the HIV-1 virus, which is a human pathogen virus that has been fully studied.
  • HIV-1 is a human pathogen virus that has been fully studied.
  • HIV-2 Simian immunodeficiency virus (SIV), or non-primate
  • Animal-like lentiviruses include Feline immunodeficiency virus (FIV), Bovine immunodeficiency virus (BIV) or Caprine arthritis-encephalitis virus (CAEV).
  • FMV Feline immunodeficiency virus
  • BIV Bovine immunodeficiency virus
  • CAEV Caprine arthritis-encephalitis virus
  • Vectors based only on Equine infectious anemia virus Equine infectious anemia virus, EIAV
  • EIAV Equine infectious anemia virus
  • the first-generation lentiviral vector system is represented by the three-plasmid system, which consists of packaging plasmids, envelope plasmids, and transfer vector plasmids containing viral genome transcription cassettes (transcriptional casstte) carrying target nucleic acid fragments. Plasmid composition.
  • the packaging plasmid is derived from the HIV-1 proviral genome. Its 5'-end LTR is replaced by the cytomegalovirus early promoter, and the 3'LTR is replaced by the simian virus-40 polyadenylic acid (SV40 polyA) sequence, and HIV-1 is deleted.
  • This packaging plasmid simultaneously expresses rev, vif, vpr, vpu and nef accessory genes.
  • the deleted HIV-1 envelope gene was replaced with the envelope protein gene VSV-G of Vesicular Stomatitis virus (Vesicular stomatitis virus) and expressed by the envelope plasmid.
  • the transfer vector plasmid carrying the viral genome transcription cassette carrying the target nucleic acid fragment carries the 5'-end LTR of HIV-1, and all 5'-end untranslated regions, the 5'-end gag gene of about 300bp, and the central polypurine tract (cppt ) Fragment, in addition to the rev response element (RRE) fragment.
  • This transfer vector plasmid is used to clone the target nucleic acid fragment and provide viral genomic RNA when the virus is assembled.
  • the second-generation lentiviral vector system is improved on the basis of the first-generation. It deletes all HIV-1 auxiliary genes (vif, vpr, vpu and nef genes) in the packaging plasmid. The removal of these auxiliary genes does not affect the titer and infectious ability of the virus, and at the same time increases the safety of the vector.
  • the third-generation lentiviral vector system consists of four plasmids, which remove the rev gene from the packaging plasmid and place it separately on another packaging plasmid.
  • the third-generation lentiviral vector system adds two safety features at the same time: the first safety feature is to construct a self-inactivating lentiviral transfer vector plasmid, that is, to delete the U3 region of the 3′ LTR in the viral genome transcription cassette, so that The lentiviral vector permanently loses the U3 region enhancer and promoter fragments of the 5'LTR and 3'LTR after the reverse transcription reaction is completed, so that even if all the viral proteins are present at this time, the viral genomic RNA can no longer be transcribed. The virus cannot be packaged successfully, so the third-generation transfer vector plasmid is also called self-inactivating (SIN) transfer vector plasmid.
  • SI self-inactivating
  • the second safety feature is that the tat gene with transcriptional transactivation function is removed, and the U3 region enhancer and promoter sequence of 5'LTR is replaced with a heterologous promoter sequence to transcribe viral genomic RNA, and when transcribing lentiviral genomic RNA , The heterologous promoter itself cannot be transcribed, so it further ensures that the lentiviral vector can only be packaged and transfected once.
  • the third-generation lentiviral system only retains the gag, pol and rev gene sequences in the original HIV-1 genome, so the third-generation lentiviral vector system is safer.
  • Retroviral/lentiviral vectors can have different pseudotypes by replacing different heterologous envelope glycoproteins, such as Lentivirus, such as human, ape, cat, and bovine immunodeficiency virus (immunodeficiency virus). ), caprine arthritis-encephalitis virus (caprine arthritis-encephalitis virus), equine infectious anemia virus (Equine infectious anemia virus, etc.) envelope protein, retrovirus (Retroviruse, such as Murine leukemia virus (10A1) ,4070A), Gibbon ape leukemia virus, Feline leukemia virus (RD114), Amphotropic retrovirus, Ecotropic retrovirus, Baboon ape leukemia virus ( Baboon ape leukemia virus, etc.) envelope protein, Paramyxovirus (Paramyxoviruses, such as Measles virus, Nipah virus, etc.) envelope protein, Rhabdoviruses, such as Rabies virus (Rabies virus) ), Mokola virus (Moko
  • retroviral/lentiviral vectors are preferably prepared using mammalian cell lines as host cells, and the most widely used are 293T or HEK293 cell lines and derived cell lines selected or modified based on them.
  • HEK293 cells are human renal epithelial cell lines transfected with adenovirus E1A gene.
  • 293T cells are derived from HEK293 cells and express SV40 large T antigen at the same time.
  • the plasmid containing the SV40 replication origin and promoter region can be replicated in 293T cells. Maintain a high plasmid copy number within a period of time, and increase the protein expression of the gene carried by the plasmid.
  • the production/packaging cells that can be used for viral vectors also include mammalian cells HepG2 cells, CHO cells, BHK cells, COS cells, NIH/3T3 cells, Vero cells, HT1080 cells, Te671 cells, CEM cells, and NSO cells. Cells and PerC6 cells.
  • the current preparation method of retroviral/lentiviral vectors is generally in mammalian host cells, usually cells derived from mice or humans, and transfer the gag gene, pol gene, and rev gene respectively (used for lentiviral vectors) , Envelope glycoprotein genes, and viral genome transcription cassettes carrying target nucleic acid fragments (including promoters for transcription and packaging into the RNA genome of retrovirus/lentivirus; retrovirus/lentivirus genome packaging and transfection required
  • a cis-acting sequence such as 5'LTR, PBS, ⁇ packaging signal, cppt (for lentiviral vectors), RRE (for lentiviral vectors), ppt and 3'LTR sequences, etc.; target nucleic acid fragments to be transduced , 3'end polyadenylic acid signal, etc.).
  • a construct containing the above-mentioned gene such as a plasmid
  • Transient production utilizes a transfection plasmid method to introduce viral gene constructs, which uses cationic reagents that can form a complex with negatively charged DNA, allowing it to be taken up by cells via endocytosis.
  • Polyethyleneimine (PEI) is one of the most widely used and most efficient cationic reagents. At present, the PEI transfection method is mostly used clinically and industrially to introduce the above constructs, but the process is unstable after the process is scaled up. , The main problems such as low toxin production titer.
  • adenovirus to prepare retroviral/lentiviral vector is complicated and the final transfection titer is not high.
  • continuous flow electroporation technology has also been developed for large-volume cell transfection. In principle, it can also be used for transient transfection production of retrovirus/lentivirus, but this technology is still limited by its ability to scale up. And expensive equipment and consumables.
  • gag, pol, rev (used in lentiviral vectors) and env genes are firstly transferred into cells simultaneously or sequentially, and through corresponding resistance screening and cloning selection, cells are screened for stably inserted into the genome and can be expressed in high co-expression.
  • a packaging cell line packaging cell line
  • a viral genome transcription cassette carrying the target nucleic acid fragment is introduced to construct a viral vector production cell line carrying the target nucleic acid fragment.
  • non-SIN self-inactivating
  • a very effective method is to first insert a lentiviral genome transcription cassette containing a (selectable) marker gene into the original cell, and use the marker gene to screen for stable integration and high-level long-term expression of the viral vector production cell line, and then pass Site-specific recombiase (site-specific recombiase), such as FLP-FRT or Cre-lox recombinase system replaces the target nucleic acid fragment to construct the marker gene of the above-mentioned production cell line to quickly construct and stably produce the viral vector carrying the target nucleic acid fragment Production cell line.
  • Site-specific recombiase site-specific recombiase
  • the stable production cell line production process is more stable and can provide a fully characterized production platform to produce safer viral vectors with low batch-to-batch differences; secondly, this process is easier to scale up and will not be as random as the instantaneous production system.
  • the increase in culture volume leads to a rapid decrease in production titer; in addition, since no raw materials such as DNA plasmids and transfection reagents are needed, there is no need to establish an additional GMP production line for plasmid production; finally, it has a higher unit yield and a simpler production process quality control.
  • the production process based on stable production cell lines will further highlight its advantages in R&D, production, management, operation and maintenance, and cost. These advantages are beneficial to the promotion of technology and drug industrialization in the fields of gene therapy and cell therapy.
  • virus preparation is usually characterized by a low infectious particle/physical particle ratio (less than 1:100); at the same time, these viral vectors are very sensitive and lose their infectivity quickly in the cell culture supernatant at 37°C, with a half-life of about 8 -12 hours, which further increases the demand for virus capacity.
  • the final stable cell line or the toxin production titer is not high or there is high leakage (leak) expression, and high leakage expression will
  • the production cell line is unstable and the cytotoxicity caused by the leaked expression of the packaging gene during cell culture expansion affects the cell growth quality, and it is difficult to meet the process amplification requirements.
  • the present disclosure uses the Sleeping Beauty (SB) transposon system and the PiggyBac (PB) transposon system to package the gag, pol, rev (used for lentiviral vectors), env genes and nucleic acid fragments that are required for virus packaging.
  • the viral genome transcription cassette is stably inserted into the genome of host cells (e.g., 293T cells) and combined with an inducible expression system (e.g., Tet-On system and/or Cumate system) to develop an inducible and regulated virus production system, which solves the above problems .
  • target nucleic acid fragment When using a retrovirus/lentiviral vector for transduction, the target nucleic acid fragment needs to be loaded into the RNA genome packaged into the retrovirus/lentivirus.
  • the term "target nucleic acid fragment” generally can refer to a gene, such as a nucleic acid sequence encoding a protein, according to the application purpose; it can be a functional ribonucleic acid (RNA), such as small interfering RNA (siRNA for short), long chain Non-coding ribonucleic acid (long non-doding RNAs, LncRNA for short), guide RNA (gRNA) for CRISPR gene editing system, transfer RNA (tRNA), ribosomal RNA (rRNA for short) ) Or other functional ribonucleic acid coding sequences; it can be other functional nucleic acid sequences, such as homologous recombination sequences, DNA or RNA sequences that can bind to proteins, DNA or DNA that can bind to other nucleic acid
  • the RNA genome of retrovirus/or lentivirus refers to the ribonucleic acid fragment that can be packaged into the virus when constructing the retrovirus/lentiviral vector. It generally contains the necessary sequences for virus packaging and transduction such as ⁇ packaging signal, long terminal repeat Sequence (long terminal repeat, LTR).
  • the lentiviral RNA genome generally also contains all 5'untranslated regions, a 5'gag gene of about 300bp, a central polypurine tract (cppt) fragment, and a rev response element (RRE) fragment; a fragment is missing Or more than one fragment may seriously affect the packaging or transduction efficiency of the lentivirus.
  • the viral genome RNA fragments carrying the target nucleic acid fragments are generally realized by constructing a viral genome transcription cassette (transcripiontal cassette) carrying the target nucleic acid fragments.
  • the transcription cassette contains a nucleic acid sequence with a promoter function ( It can be a retrovirus/lentivirus itself (LTR sequence or other heterologous promoters), a DNA sequence corresponding to the viral RNA genome, and usually a polyadenylation signal sequence that regulates the termination of transcription.
  • a construct carrying a viral genome transcription cassette carrying a nucleic acid fragment of interest (such as constructing the transcription cassette into a transfer vector plasmid) is delivered to the host cell and then transcribed by the host cell.
  • retroviruses include, but are not limited to, lentiviruses, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse breast tumor virus (MMTV), Rous Sarcoma Virus (RSV), Fujinami Sarcoma Virus (FuSV), Moloney Murine Leukemia Virus (Mo-MLV), FBR Murine Osteosarcoma Virus (FBR MSV), Moloney Murine Sarcoma Virus (Mo-MSV), Abelson Murine Leukemia Virus (A -MLV), Avian Myeloidosis Virus-29 (MC29), and Avian Encephalomyelitis Virus (AEV), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), Bovine immunodeficiency virus (BIV), caprine arthritis-encephalitis virus (CAEV), Gibbon ape leukemia
  • MMV
  • Transposon refers to a DNA sequence that can change its position in the genome.
  • Transposons can produce or reverse mutations and change the size of the cell's genome.
  • DNA transposons can be translocated from one DNA site to another in a simple cut-and-paste manner under the action of the expressed transposase (transpotase).
  • Transposition is a precise process in which the defined DNA fragments are usually the direct repeat (DR) at both ends of the transposon, the invert repeat (IR) connected to them, and the intermediate Insert sequence (IS for short) is cut out from a DNA molecule and moved to the same or different DNA molecule or another site in the genome.
  • DR direct repeat
  • IR invert repeat
  • IS intermediate Insert sequence
  • the "Sleeping Beauty (SB) transposon system” is composed of SB transposase and transposon, which can insert specific DNA insertion sequences into the genome of vertebrates.
  • SB transposase can insert the transposon into the TA dinucleotide base pair in the recipient DNA sequence.
  • the insertion site can be located in another position of the same DNA molecule (or chromosome) or in another DNA molecule (or chromosome).
  • the SB transposon consists of the target insertion sequence and the IR/DR sequence (it contains short direct repeats (DR) inverted repeats (IR)) at its two ends for recognition by SB transposase.
  • the transposase can be encoded within the transposon, or the transposase can be provided by another source. Wild-type and different variants of transposase, IR/DR sequences and transposons in the SB transposon system are known in the art.
  • the published plasmids containing Sleeping Beauty transposon include: pT/HB (Addgene, #26555), pT2/HB (Addgene, #26557), pT3 (Yant, SR, et al. (2004). "Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase: critical residues for DNA binding and hyperactivity in mammalian cells. "Mol Cell Biol 24(20): 9239-9247.) etc.
  • Variants of SB transposase include, but are not limited to, SB10, SB11, SB100X (the coding sequence is shown in SEQ ID NO: 30), etc.
  • SB transposon system may include SB transposon systems containing wild-type and different variants of SB transposase and wild-type and different variants of SB transposons.
  • the "PiggyBac(PB) transposon system" derived from Trichoplusia ni is composed of PB transposase and transposon, which can effectively transpose between the vector and the chromosome through a cut-and-paste mechanism .
  • PB transposase recognizes the transposon-specific inverted terminal repeat (Inverted terminal repeart, referred to as ITR) located at both ends of the transposon vector.
  • ITR transposon-specific inverted terminal repeat
  • 3'ITR such as SEQ ID NO: 24 or its complement
  • 5'ITR such as SEQ ID NO: 25 or the sequence shown in its complementary sequence
  • the powerful activity of the PiggyBac transposon system allows the target insertion sequence between two ITRs in the PB transposon vector to be easily moved to the target genome.
  • the transposase in the PB transposon system and wild-type and different variants of the transposon (such as ePiggyBac, the coding sequence is shown in SEQ ID NO: 31) are known in the art.
  • PB transposon system may include PB transposon systems containing wild-type and different variants of PB transposase and wild-type and different variants of PB transposons.
  • the inducible expression system is used to control the transcription and expression of the relevant genes in the nucleic acid construct transferred into the host cell.
  • examples of usable induction expression systems include, but are not limited to, the Tet-Off induction expression system in the tetracycline induction system (see, for example, Gossen, M. and H. Bujard (1992).
  • the inducible expression system for inducible expression is the Tet-On inducible expression system or the Cumate inducible expression system.
  • the inducible expression system for inducible expression is a combination of the Tet-On inducible expression system and the Cumate inducible expression system.
  • the reverse tetracycline controlled transactivator rtTA
  • Tet-On in the Tet-On inducible expression system, the reverse tetracycline controlled transactivator (rtTA) can only be combined with Tet-On in the presence of tetracycline derivatives such as tetracycline or doxycycline (Dox).
  • the expression system TRE response element (a nucleic acid sequence containing multiple copies of a continuous TetO operator sequence and a minimal promoter sequence, Tet Response Element, hereinafter referred to as TRE) is used to initiate the transcription of the regulated nucleic acid fragments connected downstream.
  • Tet-On inducible expression systems include the second-generation Tet-On Inducible Expression System (Tet-On Advanced, Clontech, in which the transactivator rtTA and the response element TRE are respectively referred to as rtTA adv (encoding nucleic acid sequence: SEQ ID NO: 29) and TRE adv (SEQ ID NO: 21) and the third generation Tet-On inducible expression system (Tet-On3G, Clontech, in which the transactivator rtTA and the response element TRE are respectively referred to as rtTA hereinafter 3G (coding nucleic acid sequence: SEQ ID NO: 28) and TRE 3G (SEQ ID NO: 19).
  • the transactivators rtTA and TRE in the second and third generation Tet-On inducible expression systems can be used in combination, for example rtTA adv can be used in combination with TRE 3G , and rtTA 3G can also be used in combination with TRE adv .
  • Cumate inducible expression system (Cumate Inducible System) was developed from Pseudomonas putida's p-cym operon, which consists of CuO operator and CuO connected downstream of the promoter TATA box Cumate and its derivatives rely on the repressor CymR protein composition that can bind to the CuO operator.
  • the CymR protein binds to the CuO operator to inhibit the transcription and expression of the target nucleic acid sequence linked downstream; when the CymR protein and After Cumate is combined, its affinity with the CuO operator decreases and separation no longer inhibits the transcriptional expression of the downstream linked target nucleic acid sequence.
  • the Tet-On inducible expression system and the Cumate inducible expression system are used in combination , Constructed a composite response element sequence containing multiple copies of the continuous TetO operator sequence, the minimal promoter sequence containing the TATA box, and the CuO operator sequence.
  • inducible expression systems that can be used for the purpose of the present disclosure may also include metal ion inducible, hormone inducible, and temperature inducible inducible expression systems.
  • the gag and pol genes (gag/pol in the examples indicate that the gag gene and the pol gene are located in the same construct, and are translated and expressed by a frameshift method), rev genes, and viral envelope proteins
  • the transcription of one or more of the coding sequence (for example VSV-G) and the viral genome transcription cassette carrying the nucleic acid fragment of interest is controlled, and the controlled transcription is induced by placing the gene or sequence in It is achieved under the control of an expression system, preferably, the controlled transcription is achieved by placing the gene or sequence under the control of the Tet-On and/or Cumate inducible expression system, more preferably in the Tet-On Under the dual control of Cumate and Cumate-induced expression or under the sole regulation of the third-generation Tet-On inducible expression system.
  • the transcription of gag and pol genes, rev genes, viral envelope protein coding sequences, and viral genome transcription cassettes carrying target nucleic acid fragments are all controlled, and the controlled transcription is performed by Placing the gene or sequence under the control of an inducible expression system, preferably, the controlled transcription is realized by placing the gene or sequence under the control of a Tet-On and/or Cumate inducible expression system It is more preferably under the dual control of the Tet-On and Cumate inducible expression system or under the sole control of the third-generation Tet-On inducible expression system.
  • the gag and pol genes, the rev gene, the coding sequence of the viral envelope protein (VSV-G) and the rev gene and the viral envelope protein (VSV-G) in the viral genome transcription cassette carrying the nucleic acid fragment of interest The transcription of the coding sequence of G) is controlled.
  • the controlled transcription is achieved by placing the gene or sequence under the control of an inducible expression system.
  • the controlled transcription is achieved by placing the gene or sequence under the control of an inducible expression system.
  • the gene or sequence is implemented under the control of Tet-On and/or Cumate inducible expression system, more preferably under the dual control of Tet-On and Cumate inducible expression or in the third-generation Tet-On inducible expression system Under individual regulation.
  • the expression level of the regulated nucleic acid fragment under inducing conditions and the leaked expression level under non-inducing conditions are two important indicators for evaluating the pros and cons of the induced expression system.
  • a good induced expression system needs to have a higher induced expression level and a lower expression level. Leak the amount of expression.
  • the present disclosure designs and uses four response elements, TRE adv , TRE 3G , TRE adv CuO, and TRE 3G CuO, and two transactivators , rtTA adv and rtTA 3G.
  • the third-generation TRE 3G response element is more stringent than the second-generation TRE adv response element, and the regulated nucleic acid fragment has a lower leakage expression, but the induced expression is lower than TRE adv .
  • the Tet-On and Cumate composite induction expression system response elements TRE adv CuO (SEQ ID NO: 22) and TRE 3G CuO (SEQ ID NO: 20) designed in the present disclosure are more stringent than any single Tet-On induction system response element. Significantly reduces the leakage expression of the regulated nucleic acid fragment, but at the same time, it also significantly reduces the induced expression of the regulated nucleic acid fragment.
  • the transactivator rtTA 3G has similar transcriptional activation activity under inducing conditions; under non-inducing conditions, the basic leakage transcriptional activation activity of rtTA 3G is lower.
  • the intron connected to the 3'end of the promoter in the expression plasmid can generally increase the stability of transcribed messenger RNA (mRNA) and increase the efficiency of mRNA transport out of the nucleus (see Akef, A., et al. (2015).” Splicing promotes the nuclear export of beta-globin mRNA by overcoming nuclear retention elements. "RNA 21(11):1908-1920) and increases the expression of regulated nucleic acid fragments.
  • the present inventors found that connecting a cleavable intron between the 3'end of the response element and the 5'end of the regulated nucleic acid fragment in a single inducible expression system can increase the inducible expression of the regulated nucleic acid fragment, but it will also Significantly increase the amount of leaked expression of regulated nucleic acid fragments.
  • the insertion of a cleavable intron sequence between the 3'end of the response element and the 5'end of the regulated nucleic acid fragment can leak expression without significantly increasing and without induction. At the same time, the amount of inducible expression under inducing conditions is significantly increased.
  • gag and pol genes In the lentiviral rev gene, VSV-G gene, gag and pol gene that needs to be expressed, and the viral genome transcription cassette carrying the target nucleic acid fragment, rev and the membrane protein VSV-G have obvious cytotoxicity, and rev also regulates and usually contains RRE sequences
  • the expression of gag and pol genes must be strictly controlled by an inducible expression system.
  • the expression of Gag and pol genes does not have to be strictly regulated under the condition of rev controlled, but using an inducible expression system to control the expression of gag and pol genes may further reduce the titers of viruses that leak expression under non-inducible conditions, especially in single Tet- The On induced expression system controls the expression of rev and VSV-G.
  • TRE adv the induction system response element is the TRE adv sequence (SEQ ID NO: 21), and there is no intron between the 3'end of the response element and the 5'end of the regulated nucleic acid fragment
  • TRE adv CuO the induction system response element Is the TRE adv CuO sequence (SEQ ID NO: 22), and there is no intron between the 3'end of the response element and the 5'end of the regulated nucleic acid fragment
  • TRE 3G the induction system response element is the TRE 3G sequence (SEQ ID NO: 19), and there is no intron between the 3'end of the response element and the 5'end of the regulated nucleic acid fragment
  • TRE 3G CuO the induction system response element
  • the intron between the 3'end of the response element and the 5'end of the regulated nucleic acid fragment controlled by the response element may be directly connected to the response element and the regulated nucleic acid fragment controlled by the response element , It may not be directly connected, which can be adjusted by those skilled in the art according to the content of the present disclosure and as needed, and in the case of not directly connected, those skilled in the art can understand that the response element and the response element controlled by the response element The distance between the regulatory nucleic acid fragments should not affect the response element's control of the transcription of the regulated nucleic acid fragment.
  • the regulatory sequence of rev is preferably TRE 3G ;
  • the regulatory sequence of VSV-G is preferably TRE 3G -intron;
  • the regulatory sequences of gag and pol are preferred CMV promoter (or other eukaryotic promoters commonly used in the art) -intron or TRE 3G -intron, and more preferably TRE 3G -intron;
  • rtTA transactivator is preferably rtTA 3G .
  • transient transfection of the above-mentioned related plasmids can achieve 6.94E+05 RLU and 176 times the induced/leaked toxin production titer ratio; compared with the second-generation Tet-On system (4.64E+05 RLU, 7-fold induction/ The leaked toxin production titer ratio) increased by 49.6% in the toxin production titer, and increased by 25 times in the control of the leaked virus expression.
  • the regulatory sequence of rev is preferably TRE adv CuO, TRE adv CuO-intron, TRE 3G , TRE 3G CuO, and TRE 3G CuO -Introns, more preferably TRE 3G , TRE 3G CuO and TRE 3G CuO-introns, and still more preferably TRE 3G ;
  • the regulatory sequence of VSV-G is preferably TRE adv CuO, TRE adv CuO-intron, TRE 3G- Introns and TRE 3G CuO-introns, more preferably TRE adv CuO-introns, TRE 3G -introns and TRE 3G CuO-introns, still more preferably TRE 3G CuO-introns; gag and
  • the regulatory sequence of pol is preferably a regulatory sequence with introns, including CMV promoter (or other e
  • transient transfection of the above-mentioned related plasmids can achieve 1.46E+06 RLU and 12032-fold induced/leaked toxin production titers; compared to the second-generation Tet-On system (4.64E+05 RLU, 7-fold induced/leaked toxin production)
  • the leaked toxin production titer ratio increased by 214.7% in the toxin production titer, and increased by 1,719 times in the control of the leaked virus expression.
  • RNA splicing can be performed in mammalian cells. (Splicing) sequence can achieve the above functions.
  • Intron sequences that can be selected include, but are not limited to, introns on commonly used cloning vectors, such as rabbit ⁇ -globulin introns, hybrids derived from human ⁇ -globulin and immunoglobulin heavy chain introns Intron, EF-1 ⁇ intron A, SV40 intron, hybrid intron derived from adenovirus and immunoglobulin heavy chain intron, modified human cytomegalovirus intron, from chicken
  • the heterozygous introns of ⁇ -actin (CBA) and mouse parvovirus (MMV) introns, chimeras derived from chicken ⁇ -actin and rabbit ⁇ -globulin introns and mP1 Intron it can also be any intron of any gene in any eukaryote; or an artificial intron sequence designed based on intron splicing rules.
  • the intron sequence used in the embodiments of the present disclosure may be, for example, the human ⁇ -globulin intron sequence (BGI sequence)
  • a transposon system is used to stably integrate gag and pol genes, rev genes, viral envelope protein (VSV-G) coding sequences, and viral genome transcription cassettes carrying target nucleic acid fragments into the host cell genome.
  • VSV-G viral envelope protein
  • viral genome transcription cassettes carrying target nucleic acid fragments when one or more of gag and pol genes, rev genes, viral envelope protein (VSV-G) coding sequences and/or viral genome transcription cassettes carrying target nucleic acid fragments are placed in Tet Under the control of -On and/or Cumate inducible expression system, use the transposon system to transfer gag and pol genes, rev genes, viral envelope protein (VSV-G) coding sequences and viral genome transcription cassettes carrying target nucleic acid fragments And the coding sequence of Tet-On transactivator rtTA protein and/or the coding sequence of Cumate operon repressor CymR protein are integrated into the host cell genome.
  • the gag and pol genes, rev genes, viral envelope protein (VSV-G) coding sequences, and viral genome transcription cassettes carrying target nucleic acid fragments are combined using the SB transposon system or the PB transposon system
  • the inducing expression system activator or repressor protein coding sequence for example, Tet-On transactivator protein coding sequence and/or Cumate operon repressor protein CymR coding sequence
  • the SB transposon system and the PB transposon system are used to transfer the above-mentioned genes and sequences into host cells, respectively.
  • gag and pol genes, rev genes, viral envelope protein (VSV-G) coding sequences and viral genome transcription cassettes carrying target nucleic acid fragments and, using Tet-On and/or In the case of Cumate-induced expression system, the coding sequence of the Tet-On transactivator protein and/or the coding sequence of the Cumate operon repressor CymR protein) is integrated into the host cell genome twice.
  • VSV-G viral envelope protein
  • any one of the SB transposon system or the PB transposon system to combine the coding sequences of gag and pol genes, rev genes, viral envelope proteins (VSV-G) and nucleic acid fragments carrying the target
  • the viral genome transcription cassette (and, in the case of the Tet-On and/or Cumate induction expression system, the coding sequence of the Tet-On transactivator protein and/or the coding sequence of the CymR protein, the repressor of the Cumate operon)
  • One or more of them are integrated into the host cell genome to construct a packaging cell line, and another unused transposon system in the SB transposon system or the PB transposon system is used for the second time to transfer the gag and pol genes , Rev gene, viral envelope protein (VSV-G) coding sequence and viral genome transcription cassette carrying the target nucleic acid fragment (and, in the case of using Tet-On and/or Cumate inducible expression system, Tet-On trans The remaining one or more of the coding sequence of the activ
  • any one of the SB transposon system or the PB transposon system to encode gag and pol genes, rev genes, and viral envelope protein (VSV-G) Sequence (preferably, and the coding sequence of the inducible expression system activator and/or repressor protein, for example, in the case of using Tet-On and/or Cumate to induce the expression system, the coding sequence of the Tet-On transactivator protein and / Or the coding sequence of the CymR protein, the repressor of the Cumate operon) is integrated into the host cell genome to construct a packaging cell line, and then another unused transposon system in the SB transposon system or the PB transposon system is used Then the viral genome transcription cassette carrying the nucleic acid fragment of interest is integrated into the genome that has integrated gag and pol genes, rev genes and viral envelope protein (VSV-G) coding sequences (preferably, as well as inducible expression system activators and / Or repressor protein coding sequence,
  • VSV-G viral envelope protein
  • stable packaging cells are transferred into a single viral genome transcription cassette construct carrying the nucleic acid fragment of interest, and simple screening is carried out.
  • the cell clone that stably integrates the above construct in the genome can be constructed and used for Preparation of a lentiviral vector production cell line carrying a lentiviral vector of the target nucleic acid fragment.
  • the inventors of the present invention found that the use of different transposon systems when integrating one or more genes or sequences into the host cell genome twice can effectively avoid the occurrence of the above-mentioned adverse effects. Therefore, in the present disclosure, different transposon systems are used during the two integrations of one or more genes or sequences into the host cell genome, that is, for example, the SB system is first used to transfer the above-mentioned one or more genes.
  • the sequence is integrated into the host cell genome, and then the PB system is used to integrate the remaining one or more genes or sequences into the host cell genome, or the PB system is first used to integrate the above one or more genes or sequences into the host cell In the genome, the SB system is then used to integrate the remaining one or more genes or sequences into the host cell genome.
  • the SB system first use the SB system to encode the gag and pol genes, rev genes and viral envelope protein (VSV-G) coding sequences (preferably, as well as the inducible expression system activator and/or repressor protein coding Sequence, for example, in the case of using Tet-On and/or Cumate inducing expression system, the coding sequence of Tet-On transactivator protein and/or the coding sequence of CymR protein, the repressor of Cumate operon) is integrated into the host cell Construct packaging cells in the genome, and then use the PB system to integrate the viral genome transcription cassette carrying the target nucleic acid fragment into the genome.
  • VSV-G viral envelope protein
  • gag and pol genes, rev genes, and viral envelope protein have been stably integrated in the genome.
  • the coding sequence of the activator and/or repressor protein of the inducing expression system for example, in the case of using Tet-On and/or Cumate to induce the expression system, the coding sequence of the Tet-On transactivator protein and/or Or the coding sequence of the CymR protein, which is the repressor of the Cumate operon, to construct the production cell in the genome of the packaging cell.
  • both PB and SB transposon systems have the function of actively integrating inserted sequence fragments into the host cell genome.
  • a single transient transfection can achieve an average successful stable integration efficiency of more than 40% in the cell population under non-resistant screening conditions. It can obtain stable integration of more than 20 copies of inserted sequence fragments in the cell genome in a single transfection operation.
  • Cell clones, and multiple copies of inserts of different sizes and sequences can be integrated into the genome of the cell in a single operation.
  • the efficiency of using the PB and SB transposon systems to integrate the insert sequence fragment into the host cell genome and the total transposon construct and transposase construct when each transposon construct is introduced into the cell The molar ratio, the amount of transposon construct introduced and the length of the inserted sequence fragment in the transposon are related.
  • the construct of transposon can be DNA plasmid, minicircle DNA, linear DNA fragment or viral vector; the construct of transposase can be DNA plasmid, minicircle DNA, linear DNA Fragment, viral vector, RNA or protein.
  • DNA plasmids When DNA plasmids are used as constructs for transposons and transposases, increasing the molar ratio of transposon/transposase plasmids to 5:1 or above during transient transfection can significantly increase the insertion sequence fragments in the cell genome The stable integration efficiency. In addition, the integration efficiency of the transposon in the cell genome decreases with the increase of the inserted sequence fragments, and there is a relatively stable linear relationship between the two. In the operation of integrating multiple inserted sequence fragments in the cell genome at the same time, you can insert the sequence according to the transposon. The length of the fragments adjusts the added amount and relative molar ratio of each transposon construct to control the copy number and molar ratio of the final inserted sequence fragments integrated into the cell genome.
  • the SB transposon system it is preferable to first use the SB transposon system to transfer the coding sequences of gag and pol genes, rev genes and viral envelope protein (VSV-G) (preferably, as well as the inducible expression system activator and/or Repressor protein coding sequence, for example, in the case of using Tet-On and/or Cumate inducing expression system, Tet-On transactivator protein coding sequence and/or Cumate operon repressor CymR protein coding sequence) Integration into the host cell genome.
  • VSV-G viral envelope protein
  • the molar ratio of the total transposon plasmid and the SB transposase plasmid is more than 5:1, more preferably
  • the above-mentioned packaging cell line was constructed under the conditions of 10:1 to 40:1.
  • the constructed packaging cell line genome stably integrates at least one copy of the gag and pol genes, rev gene and viral envelope protein (VSV-G) coding sequence (preferably, and at least one copy of the inducible expression system activator and/ Or repressor protein coding sequence, for example, in the case of using Tet-On and/or Cumate inducing expression system, at least one copy of Tet-On transactivator protein coding sequence and/or at least one copy of Cumate operon
  • the repressor CymR protein coding sequence preferably adjust the number of copies of gag and pol genes integrated into the host cell genome at 2 copies per cell; the gag and pol gene sequences and the VSV-G protein coding sequence are integrated into the host cell
  • the copy ratio of the genome is between 1:1 to 3:1; it is more preferable to adjust the number of copies of gag and pol genes integrated into the host cell genome at 4 to 6 copies per cell; gag and pol gene sequences and VSV-G
  • the copy ratio of protein coding sequence integrated into host cell genome is
  • the PB transposon system is used to integrate the viral genome transcription cassette carrying the target nucleic acid fragment into the genome of the packaging cell line.
  • the transposon plasmid and the PB transposase plasmid containing the viral genome transcription cassette carrying the target nucleic acid fragment are transiently transfected, and the molar ratio of the transposon plasmid and the PB transposase plasmid is more than 5:1.
  • a stable lentivirus production cell line is constructed under the conditions of 10:1 to 40:1.
  • At least one copy of the viral genome transcription cassette carrying the nucleic acid fragment of interest is stably integrated into the genome of the constructed production cell line.
  • This method of using the PB and SB double transposon system to construct a production cell line can increase the copy number of inserted genes, accurately adjust the molar ratio of each inserted sequence fragment integrated into the host cell genome, and improve the success of constructing and screening the optimal high-producing cell line Obvious advantages such as reducing cell screening steps and screening time, reducing the number of resistance genes used.
  • the coding sequence of gag gene, pol gene, rev gene, viral envelope protein (VSV-G), viral genome transcription cassette carrying the nucleic acid fragment of interest, and inducible expression system activator and/or repressor Protein coding sequences were constructed on different transposon constructs; in the present disclosure
  • any two of the above genes or sequence fragments are constructed on one transposon construct; in one aspect of the present disclosure, any three of the above genes or sequence fragments are constructed on one transposon construct Above;
  • any four of the above genes or sequence fragments are constructed on a transposon construct; in one aspect of the present disclosure, any five of the above genes or sequence fragments are constructed on On one transposon construct; in one aspect of the present disclosure, any six of the above genes or sequence fragments are constructed on one transpos
  • gag gene and the pol gene were constructed on the same transposon construct; the rev gene, the coding sequence of the viral envelope protein (VSV-G) and the viral genome transcription cassette carrying the target nucleic acid fragment were constructed in three independent transposons.
  • the Tet-On transactivator rtTA protein coding sequence is constructed on an independent transposon construct; when using Tet-On and Cumate
  • the coding sequence of the Tet-On transactivator rtTA protein and the coding sequence of the CymR protein, the repressor of the Cumate operon are preferably constructed on the same transposon construct.
  • the constructs of the sequence for example, the coding sequence of the Tet-On transactivator rtTA protein and/or the coding sequence of the Cumate operon repressor CymR protein
  • the screening gene sequence is a screening gene sequence for eukaryotic cells.
  • the screening gene can be selected from, for example, a hygromycin resistance gene, a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, and Bleomyces Resistance gene; or the screening gene is a metabolic pathway screening gene, for example, selected from nucleic acid sequences encoding dihydrofolate reductase, glutamine synthetase, and thymidine kinase .
  • the expression of the screening gene can be regulated and expressed by a promoter, an internal ribosome entry site (IRES), or a P2A self-splicing polypeptide sequence, preferably by the SV40 promoter.
  • IRS internal ribosome entry site
  • the gag and pol genes, rev genes, and viral envelope protein (such as VSV-G) coding sequences (preferably, as well as inducible expression system activators and/or Repressor protein coding sequence, for example, in the case of using Tet-On and/or Cumate inducing expression system, Tet-On transactivator rtTA protein coding sequence and/or Cumate operon repressor CymR protein coding sequence )
  • VSV-G viral envelope protein
  • inducible expression system activators and/or Repressor protein coding sequence for example, in the case of using Tet-On and/or Cumate inducing expression system, Tet-On transactivator rtTA protein coding sequence and/or Cumate operon repressor CymR protein coding sequence
  • one or more of the constructs may carry different screening gene sequences.
  • only one screening gene sequence is used, which can be located in any of the above.
  • a construct for example, it can be located on a construct that carries an inducible expression system activator and/or repressor protein coding sequence (for example, in the case of a Tet-On inducible expression system alone, Tet-On transactivates
  • the construct of the coding sequence of the rtTA protein carries a screening gene sequence (for example, the hygromycin resistance gene (HygroR)); or in the case of using the Tet-On and Cumate complex inducible expression system, Tet-On reaction
  • the coding sequence of the formula activator rtTA protein and the coding sequence of the CymR protein, the repressor of the Cumate operon are in the same construct that carries a selection gene sequence (for example, the hygromycin resistance gene (HygroR)).
  • the transposon system stably integrates the viral genome transcription cassette carrying the target nucleic acid fragment into the above-mentioned packaging cell line genome to construct a production cell line.
  • the construct of the viral genome transcription cassette carrying the target nucleic acid fragment preferably carries and Another resistance gene sequence (for example, puromycin resistance gene (PuroR)) that was different before.
  • the construct of the coding sequence of the viral envelope protein (VSV-G) carries the same sequence as described above.
  • Two different third resistance gene sequences e.g., blasticidin resistance gene (BSD)).
  • each of the constructs may not contain any screening genes, and only stable and high-yield packaging/production cell lines can be screened through the toxin-producing ability of monoclonal cells.
  • the lentiviral genome transcription cassette carrying the target nucleic acid fragment can be derived from the transfer vector plasmid of the second-generation lentiviral vector, such as pLVPRT-tTR-KRAB (Addgene, #11648), pLenti CMVtight eGFP Puro (w771-1) ( Addgene, #26431) or third-generation lentiviral vector transfer vector plasmids such as pSLIK-Hygro (Addgene, #25737), pHIV-EGFP (Addgene, #21373), pSico (Addgene, #11578), pRRLSIN.cPPT.PGK -GFP.WPRE (Addgene, #12252), Tet-pLKO-puro (Addgene, #21915), pLenti-puro (Addgene, #39481), pLVUT-tTR-KRAB (Addgene, #11651), etc.
  • Most of the viral genome transcription cassettes of the third-generation lentiviral vector and the second-generation lentiviral vector share LTR, 5'non-coding fragments, HIV-1 ⁇ packaging signal, RRE, cPPT and gag partial sequences, etc. in virus packaging and transduction
  • the main difference between the third generation and the second generation lentiviral genome transcription cassette is that a constitutive active promoter such as CMV or RSV is used to replace the U3 sequence that functions as a promoter in the 5'LTR sequence, and The U3 sequence of the 3'-LTR sequence was deleted, making the lentiviral transfer vector a SIN (self-inactivating) vector.
  • pSLIK-Hygro, pHIV-EGFP, and pSico vectors use the CMV promoter to transcribe lentiviral genomic RNA
  • pRRLSIN.cPPT.PGK-GFP.WPRE, Tet-pLKO-puro, pLenti -Puro uses the RSV promoter to transcribe lentiviral genomic RNA.
  • Tet-pLKO-puro and pLenti-puro do not contain WPRE sequence in the lentiviral genome transcription cassette.
  • the lentiviral genome transcription cassette in the above-mentioned lentiviral transfer vector plasmid is in principle applicable to the method of constructing a stable lentivirus production cell line described in the present disclosure.
  • the sequence of the lentiviral genome transcription cassette used in the present disclosure was designed based on the nucleic acid sequence in the pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252) transfer vector plasmid and the plasmid construction containing this sequence was constructed body.
  • the retrovirus/lentivirus production cell line construction method described in the present disclosure can also construct a virus production cell line for rapid replacement of the target nucleic acid sequence and/or outer envelope protein, including but not limited to the use of site-specific recombination (A rapid replacement virus production cell line constructed by a site-specific recombination strategy.
  • Site-specific recombination also called conservative site-specific recombination, is a gene recombination in which DNA strand exchange occurs between homologous sequence fragments at least to a certain extent.
  • Many different genome modification strategies such as recombinase-mediated cassette exchange (RMCE), all rely on site-specific recombination, which is a targeted introduction of transcription cassettes or target nucleic acid fragments.
  • RMCE recombinase-mediated cassette exchange
  • Site-specific recombination generally consists of a site-specific recombiase (SSR) and a specific site sequence recognized by it and homologous recombination occurs. Based on the amino acid sequence homology and the relevance of the recombinase catalytic mechanism, most site-specific recombinases can be classified as tyrosine (Tyr) recombinase or serine (Ser) recombinase.
  • SSR site-specific recombiase
  • Common tyrosine recombinases are Cre or FLP; common serine recombinases include classic members such as gamma-delta and Tn3 decomposing enzymes, and the newly discovered ⁇ C31-, Bxb1-, and R4 integrase (integrase).
  • the specific site sequence that binds to the site-specific recombinase and undergoes homologous recombination is generally 30 to 200 bases in length, and is surrounded by two partially inverted repeated symmetric sequences that bind to the site-specific recombinase.
  • the composition of the intermediate sequence for homologous recombination is generally 30 to 200 bases in length, and is surrounded by two partially inverted repeated symmetric sequences that bind to the site-specific recombinase.
  • Cre-lox and FLP-FRT site-specific recombination systems are commonly used mature systems for recombinase-mediated expression cassette exchange.
  • Cre-lox system is composed of Cre recombinase and loxp sequence recognized by Cre;
  • FLP-FRT system is composed of flippase recombinase (FLP) and short flippase recognition target (FRT) recognized by FLP.
  • site-specific recombination systems are based on the number and direction of their site-specific recombinase recognition site sequences, design strategies for nucleic acid mutations in the same or different DNA molecules and recognition sites, and can achieve the excision of target nucleic acid sequence fragments. , Insertion, translocation and reversal.
  • a site-specific recombinase recognition is usually set at both ends of the nucleic acid sequence to be replaced (it may contain a marker gene or a ligation marker gene (first marker gene)).
  • the target nucleic acid sequence to be replaced (preferably connected with a second marker gene different from the first marker gene) is generally set on a vector (such as a plasmid or viral vector), and both ends of which also have site-specific recombinase recognition sequences ( The second marker gene and the target nucleic acid sequence to be replaced are both located between the two specific recombinase recognition site sequences).
  • the vector carrying the target nucleic acid fragment to be replaced and the corresponding site-specific recombinase expression vector such as plasmids, viral vectors, RNA or site-specific recombinase proteins that carry encoding site-specific recombinase
  • the original nucleic acid fragment to be replaced is replaced with the target nucleic acid fragment to be replaced with the second marker gene under the catalysis of the site-specific recombinase.
  • the target cell line that successfully completes the recombinase-mediated expression cassette exchange can be obtained.
  • Similar strategies can be used to construct rapid replacement virus production cell lines for rapid replacement of target nucleic acid fragments and/or outer envelope proteins.
  • the original nucleic acid fragment itself may contain a marker gene or a linking marker gene (first marker gene A)
  • first marker gene B the coding sequence of the original outer envelope protein linked to the marker gene
  • both ends of the original nucleic acid fragment (which itself may contain a marker gene or are connected with a marker gene) are provided with site-specific recombinase recognition site sequences, and/or The two ends of the coding sequence of the original outer envelope protein connected with the marker gene are provided with site-specific recombinase recognition site sequences.
  • the target nucleic acid fragment to be replaced When quickly replacing the target nucleic acid fragment and/or the coding sequence of the outer envelope protein, the target nucleic acid fragment to be replaced that has a specific recombinase recognition site sequence at both ends and is connected to the marker gene (second marker gene) and / Or the coding sequence of the outer envelope protein to be replaced (the marker gene (second marker gene A) and the target nucleic acid fragment to be replaced are located between the two specific recombinase recognition site sequences, and/or the marker gene ( The second marker gene B) and the coding sequence of the outer envelope protein to be replaced are located between the two specific recombinase recognition site sequences, and the second marker gene (A and/or B) and the second marker gene (A and/or B) connected to the sequence to be replaced
  • the vector of the first marker gene (A and/or B) used in the production cell line has been constructed and the corresponding site-specific recombinase expression vector are simultaneously delivered to the above-mentione
  • the above-mentioned marker gene is, for example, a resistance gene sequence for eukaryotic cells, and the resistance gene sequence is, for example, selected from: hygromycin resistance gene, puromycin resistance gene, neomycin resistance gene, Blasticidin resistance gene, bleomycin resistance gene; or the marker gene is, for example, a metabolic pathway screening gene, and the metabolic pathway screening gene is, for example, selected from encoding dihydrofolate reductase (Dihydrofolate reductase), glutamine Glutamine synthetase (Glutamine synthetase), thymidine kinase (Thymidine kinase) gene; or the marker gene is, for example, a fluorescent protein marker gene, and the fluorescent protein marker gene is, for example, selected from encoding green fluorescent protein (EGFP) and red fluorescent protein (dsRed), cherry fluorescent protein (mcherry), cyan fluorescent protein (ECFP), yellow fluorescent protein (EYFP), and other mutant-
  • host cells that can be used to construct retrovirus/lentivirus packaging cell lines and production cell lines are mammalian cells.
  • host cells suitable for use in the present disclosure are 293T cells, HepG2 cells, CHO cells, BHK cells, HEK293 cells, COS cells, NIH/3T3 cells, Vero cells, HT1080 cells, Te671 cells, CEM cells, NSO cells or PerC6 cells, and derived cells derived from the above cells.
  • the host cell is a HEK293 cell or a cell derived from a HEK293 cell.
  • the host cell is a 293T cell.
  • production/packaging cells can be cultured adherently or in suspension. In one aspect, production/packaging cells can be cultured with or without serum addition.
  • tetracycline and its derivatives that can be used in the Tet-On inducible expression system include compounds similar in structure to tetracycline, which can be combined with the tetracycline-dependent transactivator rtTA of the present disclosure, and its binding constant Ka reaches at least 10 -6 M; preferably, its binding constant Ka reaches or is stronger than 10 -9 M.
  • the tetracycline derivative may be selected from, for example, doxycycline (Dox), anhydrotetracycline (Atc), chlortetracycline, oxytetracycline, and deoxytetracycline.
  • the Cumate functional analog combined with the repressor CymR that can be used in the Cumate induction expression system can be selected from, for example, p-ethyl benzoic acid, p-propyl benzoic acid, p-isopropyl benzoic acid, and p-isopropyl benzoic acid.
  • Cumate functional analogs such as described in US Patent No. 7,745,592.
  • Item 1 A method for preparing a production cell for producing a retroviral vector carrying a nucleic acid fragment of interest, the method comprising:
  • the Sleeping Beauty (SB) transposon system to transfer one or more of the sequences of the gag and pol genes of the retrovirus, the coding sequence of the viral envelope protein, and the viral genome transcription cassette sequence carrying the target nucleic acid fragment Species, but not all, are integrated into the genome of the host cell, and then the piggyBac (PB) transposon system is used to combine the gag and pol gene sequences of the retrovirus, the coding sequence of the viral envelope protein, and the nucleic acid fragments of interest. The remaining one or more of the viral genome transcription cassette sequence is further integrated into the genome of the host cell, or the PB transposon system is used to encode the sequences of the gag and pol genes of the retrovirus and the viral envelope protein.
  • SB Sleeping Beauty
  • Sequence, and one or more but not all of the viral genome transcription cassette sequence carrying the target nucleic acid fragment integrated into the genome of the host cell and then use the SB transposon system to transfer the gag and pol genes of the retrovirus
  • the remaining one or more of the sequence, the coding sequence of the viral envelope protein, and the viral genome transcription cassette sequence carrying the target nucleic acid fragment is further integrated into the genome of the host cell.
  • Item 2 A method for preparing production cells for producing a lentiviral vector carrying a nucleic acid fragment of interest, the method comprising:
  • the SB transposon system uses the SB transposon system to integrate one or more, but not all, of the gag, pol, and rev gene sequences of the lentivirus, the coding sequence of the viral envelope protein, and the viral genome transcription cassette sequence carrying the target nucleic acid fragment into In the genome of the host cell, the PB transposon system is then used to transfer the gag, pol, and rev genes of the lentivirus, the coding sequence of the viral envelope protein, and the remaining sequence of the viral genome transcription cassette carrying the target nucleic acid fragment.
  • One or more of them are further integrated into the genome of the host cell, or the sequence of the gag, pol and rev genes of the lentivirus, the coding sequence of the viral envelope protein, and the nucleic acid fragment carrying the target
  • One or more but not all of the transcription cassette sequence of the viral genome is integrated into the genome of the host cell, and then the sequence of the gag, pol and rev genes of the lentivirus and the viral envelope protein are combined using the SB transposon system.
  • the remaining one or more of the coding sequence of, and the viral genome transcription cassette sequence carrying the nucleic acid fragment of interest are further integrated into the genome of the host cell.
  • Item 3 The method according to item 2, wherein the sequence of the gag, pol and rev genes of the lentivirus and the coding sequence of the viral envelope protein are integrated into the genome of the host cell using the SB transposon system, Then use the PB transposon system to further integrate the viral genome transcription cassette sequence carrying the target nucleic acid fragment into the genome of the host cell, or use the PB transposon system to integrate the gag, pol and rev gene sequences of the lentivirus and the virus The coding sequence of the envelope protein is integrated into the genome of the host cell, and then the viral genome transcription cassette sequence carrying the target nucleic acid fragment is further integrated into the genome of the host cell using the SB transposon system.
  • Item 4 The method according to item 2, wherein the sequences of the gag, pol and rev genes of the lentivirus, the coding sequence of the viral envelope protein, and the viral genome transcription cassette sequence carrying the target nucleic acid fragment are located in two On the above construct.
  • Item 5 The method according to item 4, wherein the sequence of the gag and pol genes are located on one construct, the sequence of the rev gene is located on another construct, and the coding sequence of the viral envelope protein is located on the third construct And the viral genome transcription cassette sequence carrying the nucleic acid fragment of interest is located on the fourth construct.
  • Item 6 The method according to item 2, wherein the gag, pol, and rev genes of the lentivirus are the gag, pol, and rev genes of the HIV-1 virus.
  • the viral envelope protein is selected from the group consisting of feline leukemia virus (RD114) envelope protein, amphoteric retrovirus envelope protein, tropic retrovirus envelope protein, baboon Simian Leukemia Virus Envelope Protein, Nipah Virus Envelope Protein, Mokola Virus Envelope Protein, Lymphocyte Choroid Meningitis Virus Envelope Protein, Chikungunya Virus Envelope Protein, Ross River Virus Envelope Protein, Sam Niche Forest Virus Envelope Protein, Sindbis Virus Envelope Protein, Venezuelan Equine Encephalitis Virus Envelope Protein, Western Equine Encephalitis Virus Envelope Protein, Influenza Virus Envelope Protein, Avian Disease Virus Envelope Protein, Chandip Pull virus and piri virus envelope protein, simian immunodeficiency virus envelope protein, feline immunodeficiency virus envelope protein, equine infectious anemia virus envelope protein, Ebola virus envelope protein, rabies virus
  • Item 8 The method according to item 2, wherein the viral envelope protein is a glycoprotein of vesicular stomatitis virus (VSV-G).
  • VSV-G vesicular stomatitis virus
  • Item 9 The method according to item 2, wherein the transposase used in the SB system is SB100X, and/or the transposase used in the PB system is ePiggyBac.
  • Item 10 The method according to item 2, wherein the transcription of one or more of the gag, pol, and rev genes, the coding sequence of the viral envelope protein, and the viral genome transcription cassette carrying the target nucleic acid fragment is Controlled.
  • Item 11 The method according to item 10, wherein the viral envelope protein is VSV-G and the transcription of the rev gene and the coding sequence of VSV-G is controlled.
  • Item 12 The method according to item 10, wherein the viral envelope protein is VSV-G and the transcription of the gag, pol and rev genes and the coding sequence of VSV-G is controlled.
  • Item 13 The method according to Item 10, wherein the controlled transcription is achieved by placing the gene or sequence under the control of an inducible expression system.
  • Item 14 The method according to item 13, wherein the inducible expression system is selected from the group consisting of tetracycline inducible expression system and Cumate inducible expression system.
  • Item 15 The method according to item 14, wherein the gene or sequence is placed under the sole control of the Tet-On inducible expression system or under the dual control of the Tet-On and Cumate inducible expression system.
  • Item 16 The method according to Item 15, wherein the transcription of rev is under the control of the TRE 3G sequence and/or the transcription of the coding sequence of VSV-G under the sole control of the Tet-On induced expression system Under the control of TRE 3G sequence-intron, and/or the transcription of gag and pol is under the control of eukaryotic promoter-intron or TRE 3G sequence-intron; in Tet-On and Cumate inducible expression system
  • rev transcription is under the control of TRE adv CuO sequence, TRE adv CuO sequence-intron, TRE 3G sequence, TRE 3G CuO sequence or TRE 3G CuO sequence-intron, and/or VSV-
  • the transcription of G is under the control of TRE adv CuO sequence, TRE adv CuO sequence-intron, TRE 3G sequence-intron or TRE 3G CuO sequence-intron, and/or the transcription of gag and pol is initiated in eukaryotic promoter
  • Item 17 The method according to item 16, wherein, in the case of the Tet-On induced expression system alone, the transcription of rev is under the control of the TRE 3G sequence, and the transcription of the VSV-G coding sequence is under the control of the TRE 3G Under the control of sequence-intron, and the transcription of gag and pol is under the control of eukaryotic promoter-intron or TRE 3G sequence-intron; under the dual control of Tet-On and Cumate induced expression system
  • the transcription of rev is under the control of TRE 3G sequence or TRE 3G CuO sequence
  • the transcription of VSV-G is under the control of TRE 3G CuO sequence-intron
  • the transcription of gag and pol is under the control of eukaryotic promoter-intron Or TRE 3G CuO sequence-under the control of introns.
  • Item 18 The method according to item 17, wherein under the dual control of Tet-On and Cumate induced expression system, the transcription of rev is under the control of the TRE 3G sequence, and the transcription of VSV-G is under the control of the TRE 3G CuO sequence -Under the control of introns, and the transcription of gag and pol are under the control of the TRE 3G CuO sequence-introns, and the number of inserted copies of the gag/pol gene in the genome of the host cell is 2-8 copies /Cell, and the ratio of the copy number of gag/pol inserted in the genome of the host cell to VSV-G is 1:1 to 4:1.
  • Item 19 The method according to Item 18, wherein the number of inserted copies of the gag/pol gene in the genome of the host cell is 4-6 copies/cell, and the number of copies inserted in the genome of the host cell
  • the copy number ratio of gag/pol to VSV-G is 2:1 to 3:1.
  • Item 20 The method according to item 15, wherein the coding sequence of the Tet-On transactivator protein or the coding sequence of the Tet-On transactivator protein is converted by the SB transposon system or the PB transposon system
  • the coding sequence of CymR protein, which is the repressor of Cumate operon, is integrated into the genome of the host cell.
  • Item 21 The method according to Item 20, wherein the Tet-On transactivator protein is rtTA 3G .
  • Item 22 The method according to item 15, wherein the sequence of the gag, pol, and rev genes of the lentivirus, the coding sequence of the viral envelope protein, and the Tet-On transactivator protein are combined using the SB transposon system
  • the coding sequence or the coding sequence of the Tet-On transactivator protein and the coding sequence of the CymR protein, the repressor of the Cumate operon, are integrated into the genome of the host cell, and then the PB transposon system is used to transfer the virus carrying the target nucleic acid fragment
  • the genome transcription cassette sequence is further integrated into the genome of the host cell, or the sequence of the gag, pol and rev genes of the lentivirus, the coding sequence of the viral envelope protein, and the Tet-On transactivator are integrated by the PB transposon system.
  • the coding sequence of the protein or the coding sequence of the Tet-On transactivator protein and the coding sequence of the CymR protein, the repressor of the Cumate operon, are integrated into the genome of the host cell, and then the SB transposon system is used to carry the target nucleic acid fragment
  • the transcription cassette sequence of the viral genome is further integrated into the genome of the host cell.
  • Item 23 Producer cells prepared by the method according to any one of items 1-22.
  • Item 24 The production cell according to Item 23, wherein the production cell was deposited at the China Common Microbial Culture Collection Management Center under the deposit number CGMCC No. 19675 on April 13, 2020.
  • Item 25 A production cell for the production of a retroviral vector carrying a nucleic acid fragment of interest, the production cell integrating the sequence of the retrovirus gag and pol genes, and the coding sequence of the viral envelope protein in its genome , And the viral genome transcription cassette sequence carrying the target nucleic acid fragment, the sequences of the gag and pol genes, the coding sequence of the viral envelope protein, and the viral genome transcription cassette sequence carrying the target nucleic acid fragment have two ends respectively for being used by SB
  • the IR/DR sequence recognized by the transposase or the ITR sequence for recognition by the PB transposase, and the IR/DR sequence and the ITR sequence are present in the producer cell at the same time.
  • Item 26 The producer cell according to Item 25, wherein the sequences of the gag and pol genes and the coding sequence of the viral envelope protein each have IR/DR sequences for recognition by SB transposase,
  • the two ends of the viral genome transcription cassette sequence carrying the target nucleic acid fragment have ITR sequences for recognition by PB transposase, or the sequences of the gag and pol genes and the coding sequence of the viral envelope protein. It has an ITR sequence for recognition by PB transposase, and both ends of the viral genome transcription cassette sequence carrying the nucleic acid fragment of interest have IR/DR sequences for recognition by SB transposase.
  • Item 27 The producer cell according to Item 25, wherein the retrovirus is a lentivirus, and the producer cell further integrates the sequence of the rev gene of the lentivirus in its genome, and the sequence of the rev gene Both ends have IR/DR sequences for recognition by SB transposase or ITR sequences for recognition by PB transposase.
  • Item 28 The producer cell according to Item 27, wherein the retrovirus is an HIV-1 virus.
  • Item 29 The producer cell according to Item 27, wherein the sequences of the gag, pol, and rev genes and the coding sequence of the viral envelope protein each have IR/DR for recognition by SB transposase Sequence, and the viral genome transcription cassette sequence carrying the nucleic acid fragment of interest has an ITR sequence for recognition by PB transposase, or the sequence of the gag, pol and rev genes and the coding sequence of the viral envelope protein at both ends
  • the two ends of the virus have ITR sequences for recognition by PB transposase
  • the two ends of the viral genome transcription cassette sequence carrying the target nucleic acid fragment have IR/DR sequences for recognition by SB transposase.
  • Item 30 The producer cell according to Item 25, wherein the viral envelope protein is a glycoprotein of vesicular stomatitis virus (VSV-G).
  • VSV-G vesicular stomatitis virus
  • Item 31 The producer cell according to Item 23 or the producer cell according to Item 25, wherein the target nucleic acid fragment sequence has a recognition sequence for a site-specific recombinase system at both ends, and/or Both ends of the coding sequence of the outer envelope protein have recognition sequences for site-specific recombinase system.
  • Item 32 A target nucleic acid fragment and/or outer envelope protein replacement system for retroviral vectors, the system comprising: the production cell according to Item 31, a site-specific recombinase system, and a replacement system The target nucleic acid fragment and/or the coding sequence of the outer envelope protein.
  • Item 33 The system according to Item 32, wherein the site-specific recombinase system is FLP-FRT or Cre-lox recombinase system.
  • Item 34 The system according to item 32, wherein the original target nucleic acid fragment in the producer cell contains a marker gene or is connected to the marker gene and/or the original outer envelope protein coding sequence in the producer cell and The marker gene is connected, and the marker gene connected to the target nucleic acid fragment to be replaced and/or the coding sequence of the outer envelope protein is different from the original marker gene in the production cell.
  • Item 35 The system according to Item 34, wherein the marker gene is a resistance gene sequence for eukaryotic cells, a metabolic pathway screening gene, a fluorescent protein marker gene, which can be used for a reporter gene to detect a protease gene, or Any combination of it.
  • the marker gene is a resistance gene sequence for eukaryotic cells, a metabolic pathway screening gene, a fluorescent protein marker gene, which can be used for a reporter gene to detect a protease gene, or Any combination of it.
  • Item 36 The system according to Item 35, wherein the resistance gene sequence is selected from: hygromycin resistance gene, puromycin resistance gene, neomycin resistance gene, blasticidin resistance Gene, bleomycin resistance gene.
  • Item 37 The system according to Item 35, wherein the metabolic pathway screening gene is selected from encoding dihydrofolate reductase, glutamine synthetase, and thymidine kinase Gene.
  • Item 38 The system according to Item 35, wherein the fluorescent protein marker gene is selected from encoding green fluorescent protein (EGFP), red fluorescent protein (dsRed), cherry fluorescent protein (mcherry), cyan fluorescent protein (ECFP) ), yellow fluorescent protein (EYFP), and the gene sequences of other mutant-derived fluorescent proteins.
  • EGFP green fluorescent protein
  • dsRed red fluorescent protein
  • mcherry cherry fluorescent protein
  • ECFP cyan fluorescent protein
  • EYFP yellow fluorescent protein
  • Item 39 The system according to Item 35, wherein the protease gene that can be used for reporter gene detection is selected from the group consisting of encoding luciferase, ⁇ -galactosidase, and chloramphenicol. The gene sequence of chloramphenical acetyltransferase.
  • Item 40 A method for replacing the target nucleic acid fragment and/or outer envelope protein in a retrovirus producer cell, the method comprising: providing the producer cell according to item 31, and using site specificity The recombinase system replaces the original target nucleic acid fragment and/or the coding sequence of the outer envelope protein in the production cell with the target nucleic acid fragment and/or the coding sequence of the outer envelope protein to be replaced.
  • Item 41 The method according to Item 40, wherein the site-specific recombinase system is FLP-FRT or Cre-lox recombinase system.
  • Item 42 The method according to item 40, wherein the original target nucleic acid fragment in the producer cell contains a marker gene or is connected to the marker gene and/or the original outer envelope protein coding sequence in the producer cell and The marker gene is connected, and the marker gene connected to the target nucleic acid fragment to be replaced and/or the coding sequence of the outer envelope protein is different from the original marker gene in the production cell.
  • Item 43 The method according to Item 42, wherein the marker gene is a resistance gene sequence for eukaryotic cells, a metabolic pathway screening gene, a fluorescent protein marker gene, which can be used for a reporter gene to detect a protease gene, or Any combination of it.
  • the marker gene is a resistance gene sequence for eukaryotic cells, a metabolic pathway screening gene, a fluorescent protein marker gene, which can be used for a reporter gene to detect a protease gene, or Any combination of it.
  • Item 44 The method according to Item 43, wherein the resistance gene sequence is selected from: hygromycin resistance gene, puromycin resistance gene, neomycin resistance gene, blasticidin resistance Gene, bleomycin resistance gene.
  • Item 45 The method according to Item 43, wherein the metabolic pathway screening gene is selected from encoding dihydrofolate reductase, glutamine synthetase, and thymidine kinase Gene.
  • Item 46 The method according to Item 43, wherein the fluorescent protein marker gene is selected from encoding green fluorescent protein (EGFP), red fluorescent protein (dsRed), cherry fluorescent protein (mcherry), cyan fluorescent protein (ECFP) ), yellow fluorescent protein (EYFP), and the gene sequences of other mutant-derived fluorescent proteins.
  • EGFP green fluorescent protein
  • dsRed red fluorescent protein
  • mcherry cherry fluorescent protein
  • ECFP cyan fluorescent protein
  • EYFP yellow fluorescent protein
  • Item 47 The method according to Item 43, wherein the protease gene that can be used for reporter gene detection is selected from the group consisting of encoding luciferase, ⁇ -galactosidase, and chloramphenicol The gene sequence of chloramphenical acetyltransferase.
  • Item 48 A retrovirus producing cell obtained by the method for replacing a target nucleic acid fragment and/or an outer envelope protein in a retrovirus producing cell according to Item 40.
  • Item 49 A method for producing a retroviral vector carrying a nucleic acid fragment of interest, the method comprising the following steps:
  • Item 50 The method according to Item 49, wherein the inducer is tetracycline or a tetracycline derivative and/or Cumate and functional analogs thereof.
  • Item 51 The method according to Item 50, wherein the tetracycline derivative is doxycycline.
  • Item 52 A retroviral vector carrying the nucleic acid fragment of interest prepared by the method described in Item 49.
  • Item 53 The producer cell according to any one of items 23-31, the producer cell according to item 48, or the retroviral vector carrying the nucleic acid fragment of interest according to item 52 is being prepared for Use as a reagent for delivering target nucleic acid fragments to cells.
  • Item 54 A method for preparing packaging/production cells for the production of lentiviral vectors, the method comprising combining the sequences of the gag, pol and rev genes of the lentivirus and the glycoprotein of vesicular stomatitis virus (VSV -G)
  • the coding sequence is transferred into the host cell, and the transcription of one or more of the gag, pol and rev genes and the coding sequence of VSV-G is under the sole control of the Tet-On inducible expression system or Tet- Under the dual control of On and Cumate induced expression system, in the case of Tet-On induced expression system alone, the transcription of rev is under the control of the TRE 3G sequence, and/or the transcription of the VSV-G coding sequence is in the TRE 3G sequence -Under the control of introns, and/or the transcription of gag and pol are under the control of eukaryotic promoter-intron or TRE 3G sequence-intron; in the case of dual control of Tet-On and
  • Item 55 The method according to item 54, wherein, in the case of the Tet-On induced expression system alone, the transcription of rev is under the control of the TRE 3G sequence, and the transcription of the coding sequence of VSV-G is under the control of the TRE 3G Under the control of sequence-intron, and the transcription of gag and pol is under the control of eukaryotic promoter-intron or TRE 3G sequence-intron; under the dual control of Tet-On and Cumate induced expression system
  • the transcription of rev is under the control of TRE 3G sequence or TRE 3G CuO sequence
  • the transcription of VSV-G is under the control of TRE 3G CuO sequence-intron
  • the transcription of gag and pol is under the control of eukaryotic promoter-intron Or TRE 3G CuO sequence-under the control of introns.
  • Item 56 The method according to Item 54, wherein the sequences of the gag, pol and rev genes of the lentivirus and the coding sequence of VSV-G are integrated into the genome of the host cell.
  • Item 57 The method according to item 56, wherein the gag, pol and rev gene sequences of the lentivirus and the coding sequence of VSV-G are integrated into the host cell through the SB transposon system or the PB transposon system Genome.
  • Item 58 The method according to item 56, wherein in the case of dual control of Tet-On and Cumate induced expression system, the transcription of rev is under the control of the TRE 3G sequence, and the transcription of VSV-G is under the control of the TRE 3G CuO sequence -Under the control of introns, and the transcription of gag and pol are under the control of the TRE 3G CuO sequence-introns, and the number of inserted copies of the gag/pol gene in the genome of the host cell is 2-8 copies /Cell, and the ratio of the copy number of gag/pol inserted in the genome of the host cell to VSV-G is 1:1 to 4:1.
  • Item 59 The method according to Item 58, wherein the number of inserted copies of the gag/pol gene in the genome of the host cell is 4-6 copies/cell, and the number of copies inserted in the genome of the host cell
  • the copy number ratio of gag/pol to VSV-G is 2:1 to 3:1.
  • Item 60 The method according to item 56, wherein the gag, pol and rev gene sequences of the lentivirus and the coding sequence of VSV-G are integrated into the host cell through the SB transposon system or the PB transposon system
  • the viral genome transcription cassette sequence carrying the target nucleic acid fragment is further integrated into the genome of the host cell through the same transposon system, or the viral genome carrying the target nucleic acid fragment is transcribed by the method of transient transfection
  • the cassette sequence is further transferred into the host cell.
  • Item 61 A packaging/production cell for the production of a lentiviral vector, the packaging/production cell comprising the sequences of the gag, pol and rev genes of the lentivirus and the glycoprotein of vesicular stomatitis virus (VSV-G)
  • VSV-G vesicular stomatitis virus
  • the transcription of one or more of the gag, pol and rev genes and the coding sequence of VSV-G is under the sole control of the Tet-On inducible expression system or the dual Tet-On and Cumate inducible expression system Under the control, in the case of the Tet-On induced expression system alone, the transcription of rev is under the control of the TRE 3G sequence, and/or the transcription of the VSV-G coding sequence is under the control of the TRE 3G sequence-intron, And/or the transcription of gag and pol is under the control of eukaryotic promoter-intron or TRE 3G sequence-intron; under the dual control of Tet-On and
  • Item 62 The packaging/production cell for producing a lentiviral vector according to Item 61, wherein the sequence of the gag gene and/or the sequence of the pol gene and/or the sequence of the rev gene of the lentivirus and/or The coding sequence of the glycoprotein of vesicular stomatitis virus (VSV-G) is integrated in the genome of the packaging/production cell.
  • VSV-G vesicular stomatitis virus
  • Item 63 The packaging/production cell for the production of lentiviral vectors according to Item 61, wherein, under the sole control of the Tet-On inducible expression system, the transcription of rev is under the control of the TRE 3G sequence, and VSV
  • the transcription of the coding sequence of -G is under the control of TRE 3G sequence-intron
  • the transcription of gag and pol is under the control of eukaryotic promoter-intron or TRE 3G sequence-intron
  • Tet-On In the case of dual control with Cumate induced expression system, the transcription of rev is under the control of TRE 3G sequence or TRE 3G CuO sequence
  • the transcription of VSV-G is under the control of TRE 3G CuO sequence-intron. Transcription is under the control of eukaryotic promoter-intron or TRE 3G CuO sequence-intron.
  • Item 64 The packaging/production cell for the production of lentiviral vectors according to Item 61, wherein under the dual control of Tet-On and Cumate inducible expression system, the transcription of rev is under the control of the TRE 3G sequence, The transcription of VSV-G is under the control of the TRE 3G CuO sequence-intron, and the transcription of gag and pol is under the control of the TRE 3G CuO sequence-intron, and the gag/pol gene is under the control of the packaging/producing cell
  • the number of inserted copies in the genome is 2-8 copies/cell, and the ratio of the copy number of gag/pol inserted in the genome of the packaging/production cell to the copy number of VSV-G is 1:1 to 4:1.
  • Item 65 The packaging/production cell for producing lentiviral vectors according to Item 64, wherein the number of inserted copies of the gag/pol gene in the genome of the packaging/production cell is 4-6 copies/cell , And the copy number ratio of gag/pol inserted in the genome of the packaging/production cell to VSV-G is 2:1 to 3:1.
  • Item 66 The packaging/production cells used for the production of lentiviral vectors as described in Item 61, which were deposited at the China Common Microbial Species Collection Management Center on April 13, 2020 under the accession number CGMCC No.19674.
  • Item 67 A method of producing a lentiviral vector, which comprises culturing the packaging/production cell according to any one of items 61 to 66 under conditions suitable for the production of a lentiviral vector.
  • Figure 1 shows a map of some plasmids used in the examples.
  • Figure 2 shows the verification of the transposition specificity of the SB and PB transposon systems in Example 2.
  • the abscissa is the passage of the cells, and the ordinate is the EGFP-positive ratio of the co-transfected cells (Figure 2A) and the median EGFP fluorescence intensity (MFI) ( Figure 2B).
  • Fig. 3 shows an experimental flow chart for constructing a production cell stably producing a lentiviral vector in Example 2.
  • Figure 4 shows the test results of the toxin-producing ability of the lentiviral stable producer cells constructed in different combinations through the SB and PB transposon systems in Example 2, where the abscissa is the number of the constructed producer cell, and the ordinate is the detection virus transfection The RLU value of the titre Luciferase experiment.
  • Figure 5 shows the comparison of the effects of different regulatory sequences on the production of toxins by regulating a single lentivirus packaging gene in Example 3.
  • the figure shows the results of virus titer detection, the abscissa is the name of the different regulatory sequences to be tested; the ordinate is the RLU value of the Luciferase experiment for detecting the titre of virus transfection.
  • the detection results of the induced toxin production titer and the non-induced leakage titer of rev Figure 5A
  • VSV-G Figure 5B
  • gag/pol Figure 5C
  • Figure 6 shows the use of different optimized regulatory sequences in Example 3 to regulate the expression of rev, VSV-G and gag/pol and compare the induced toxin production titer and the non-induced leakage titer.
  • the figure shows the results of virus titer testing.
  • the abscissa is the combination of plasmids with different regulatory sequences, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
  • Figure 7 shows the effect of the copy number of a single gene integrated in the host cell genome in Example 4 on the cell's ability to produce toxins.
  • the abscissa represents the molar ratio of different plasmids when transfecting cells
  • the left ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer
  • the right ordinate is the average integrated copy number of the gene to be tested in the cell genome.
  • Figure 7A shows the effect of rtTA 3G- CymR fragment genome integration copy number on toxin production
  • Figure 7B shows the effect of rev fragment genome integration copy number on toxin production
  • Figure 7C shows the effect of VSV-G fragment genome integration copy number on toxin production
  • Figure 7D shows the effect of gag/pol fragment genome integration copy number on toxin production ability.
  • Figure 8 shows the effect of the different integrated copy number ratios of gag/pol and VSV-G fragments in the host cell genome on the cell's ability to produce toxins in Example 4.
  • the abscissa represents the molar ratio of different plasmids when transfecting cells
  • the left ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer
  • the right ordinate is the integrated copy of gag/pol and VSV-G fragments in the cell genome. The ratio of the number.
  • Figure 9 shows a flowchart of the construction, screening and suspension adaptation of the EuLV293T3rd lentivirus stable packaging cell line in Example 5.
  • Figure 10 shows the screening process of SB16 monoclonal ( Figure 10A) and SB28 monoclonal ( Figure 10B) and the screening results of 96-well plate, 24-well plate and 6-well plate in Example 5.
  • the abscissa is the ratio of the virus prepared by monoclonal cells to the RLU detection result of Luciferase standard (expressed as an index based on 2), and the ordinate is the number of monoclonal cells in each data interval.
  • the dotted line is the average value of the RLU ratios of all monoclonal samples, and the box is the monoclonal cell strains screened and retained based on the test results.
  • Figure 11 shows the results of using the HT1080 cell Luciferase virus titer detection method in Example 5 to detect the toxin-producing ability of a monoclonal packaging cell line adapted to suspension culture.
  • the abscissa is the number of the detected cell, and the ordinate is the RLU value of the Luciferase detection experiment corresponding to the virus transfection titer.
  • Figure 12 shows the effect of transient transfection of different lentiviral packaging gene plasmid combinations on the toxin production titer of adherent cultured high-yield monoclonal packaging cell lines.
  • the legend on the abscissa represents the transiently transfected lentivirus packaging gene combination, where the "-" sign represents no induction conditions, and "+” represents the induction conditions; the ordinate represents the RLU value of the Luciferase detection experiment corresponding to the virus transfection titer; solid line Represents the toxin production titer of the positive control by transiently transfecting 293T cells; the dotted line represents the toxin production titer of the positive control for induction of toxin production by each cell.
  • Figure 13 shows the effect of transient transfection of different lentiviral packaging gene plasmid combinations on the toxin production titer of suspension cultured high-yield monoclonal packaging cell lines.
  • the legend on the abscissa represents the transiently transfected lentivirus packaging gene combination, where the "-" sign represents no induction conditions, and "+” represents the induction conditions; the ordinate represents the RLU value of the Luciferase detection experiment corresponding to the virus transfection titer; solid line Represents the toxin production titer of the positive control by transiently transfecting 293T cells; the dotted line represents the toxin production titer of the positive control for induction of toxin production by each cell.
  • Fig. 14 shows the comparison of the toxin-producing ability of the lentivirus stable production cell line carrying different target nucleic acid fragments constructed in Example 7 and the toxin-producing ability of the transient transfection method.
  • the legend on the abscissa indicates the number of the tested transfer vector plasmid; the ordinate is the RLU value of the Luciferase detection experiment corresponding to the virus transfection titer
  • Figure 15 shows the induction and leakage of toxin production titers of the stable lentivirus production cells constructed in Example 7 under serum-free suspension culture conditions.
  • the legend "-" on the abscissa represents no induction conditions, and "+” represents the induction conditions; the ordinate is the RLU value of the Luciferase detection experiment corresponding to the virus transfection titer.
  • the molecular cloning techniques used in the following examples for example, PCR amplification of DNA fragments, restriction endonuclease digestion of DNA fragments, gel recovery of DNA fragments, T4 DNA ligase ligation of two or more DNA fragments, Methods such as transformation of ligation product competent cells, small-scale plasmid preparation and identification are all well-known techniques in the art.
  • PCR enzyme Thermo, F-530S
  • restriction endonuclease NEB
  • T4 DNA ligase Invitrogen, 15224041
  • DNA fragment gel recovery kit Omega, D2500-02
  • Plasmid small extraction kit TIANGEN, DP105-03
  • Competent cell XL-10 Gold, Hunan Fenghui Biotechnology Co., Ltd., JZ011
  • SEQ ID NO:1 to SEQ ID NO:18 The sequence was synthesized by GenScript and used in the construction of the plasmid described in the present disclosure, and the plasmid sequencing and identification were completed by Invitrogen.
  • Table 1 is the primer information for plasmid construction
  • Table 2 is the description of the composition of the elements of the sequence SEQ ID NO: 1 to SEQ ID NO: 31
  • Table 3 is the plasmid Description of each functional element
  • Table 4 is the plasmid number and corresponding name constructed in this disclosure.
  • sequence information of the functional elements used in the plasmids involved in the following examples is an example for realizing the present disclosure. Those skilled in the art can expect to replace the functional element sequences on the plasmids used in the following examples with other elements with similar biological functions.
  • Sequences can also achieve the effects described in the present disclosure, including but not limited to plasmid backbone sequences (such as replication origin, resistance genes, etc.), restriction site sequences, transposon repeat sequences, and induction system response element sequences , Insulator sequence, promoter sequence, intron sequence, polyadenylic acid signal (PolyA) sequence, gene sequence optimized for different codons, mutants of the above functional element sequences and gene sequences, and each function The cloning position, cloning sequence and cloning direction of the element sequence and gene sequence.
  • the specific plasmid construction method is as follows:
  • Plasmids 18BF007 and 18BF004 the synthetic sequence SEQ ID NO: 2 (2900bp) and sequence SEQ ID NO: 3 (1386bp) were digested with NotI and AsiSI and respectively connected to plasmid 18BF003 (sequence SEQ ID NO: 1,1893bp) ) NotI and AsiSI restriction sites to construct plasmids 18BF007 and 18BF004, respectively.
  • plasmids 18BF011 and 18BF063 The 18BF007 plasmid was digested with MluI and SphI, and the 1730bp fragment was recovered from the gel and ligated to the MluI and SphI digestion sites of the 18BF003 plasmid to construct the plasmid 18BF011.
  • the synthetic sequence SEQ ID NO: 4 (915 bp) was digested with MluI and ClaI and connected to the MluI and ClaI sites of 18BF007, replacing the CMV promoter to construct plasmid 18BF063.
  • sequence SEQ ID NO: 5 (887bp), sequence SEQ ID NO: 6 (897 bp) and sequence SEQ ID NO: 7 (852 bp) were digested with MluI and ClaI and connected to the MluI and ClaI restriction sites of the 18BF072 plasmid , Replacing the TRE 3G CuO-BGI fragment to construct plasmids 18BF071, 19BF249 and 19BF248 respectively.
  • Plasmids 18BF072, 18BF071, 19BF249 and 19BF248 were digested with BstBI, and 4147bp (18BF072), 4119bp (18BF071), 4129bp (19BF249) and 4084bp (19BF248) fragments were gel-recovered and ligated with T4 ligase to construct plasmids 19BF247 and 19BF246, respectively , 18BF070 and 18BF069.
  • plasmids 18BF068, 18BF067, 19BF245, 19BF244, 19BF243, 19BF242, 18BF066, 18BF065 and 19BF254 using pMD2.G (Addgene, #12259) as a template, C-VSVG-F (SEQ ID NO: 32) and C- VSVG-R (SEQ ID NO: 33) used primers to amplify the VSV-G gene fragment (1565bp) by PCR, and then digested with ClaI and XhoI and ligated to the ClaI and XhoI sites of 18BF063 plasmid to construct plasmid 18BF068.
  • sequence SEQ ID NO: 5 (887bp), sequence SEQ ID NO: 6 (897 bp) and sequence SEQ ID NO: 7 (852 bp) were digested with MluI and ClaI, respectively, and connected to the MluI and ClaI digestion positions of the 18BF068 plasmid Click to replace the TRE 3G CuO-BGI fragment to construct plasmids 18BF067, 19BF245 and 19BF244.
  • Plasmid 18BF068 was digested with ClaI and XhoI, and the 1550bp fragment was recovered from the gel and ligated to the ClaI and XhoI sites of plasmids 19BF247, 19BF246, 18BF070, and 18BF069, respectively, and replaced the rev gene to construct plasmids 19BF243 and 19BF242, respectively. , 18BF066 and 18BF065.
  • the synthetic sequence SEQ ID NO: 8 (887bp) was digested with SpeI and PvuII and connected to the AvrII and PmeI sites of the 18BF068 plasmid to construct the plasmid 19BF254.
  • Plasmids 18BF074, 19BF131, 19BF130, 19BF251, 19BF250, 19BF126, 19BF129, 19BF128 and 18BF076 use pMDLg/pRRE (Addgene, #12251) as template, C-RRE-F (SEQ ID NO: 36) and C- RRE-R (SEQ ID NO: 37) are primers for PCR amplification of RRE fragments (400 bp); C-GagPol-F (SEQ ID NO: 38) and C-GagPol-R (SEQ ID NO: 39) are primers for PCR amplification
  • the gag/pol gene fragment (4336bp) was increased, and then the two DNA fragments were digested with XbaI and XhoI and EcoRI and XbaI, respectively, and ligated to the EcoRI and XhoI restriction sites of the 18BF007 plasmid to construct the 18BF074 plasmid.
  • sequence SEQ ID NO: 4 (915 bp), sequence SEQ ID NO: 5 (887 bp), sequence SEQ ID NO: 6 (897 bp) and sequence SEQ ID NO: 7 (852 bp) were digested with MluI and EcoRI respectively and connected At the MluI and EcoRI restriction sites of the 18BF074 plasmid, the CMV-BGI fragment was replaced to construct the 19BF131, 19BF130, 19BF251 and 19BF250 plasmids.
  • the 18BF074, 19BF131, 19BF130 and 19BF250 plasmids were digested with BstBI, respectively, and the 8758bp (18BF074), 8494bp (18BF131), 8466bp (19BF130) and 8421bp (19BF250) fragments were gel-recovered and ligated with T4 ligase to construct the plasmid 19BF126. , 19BF129, 19BF128 and 18BF076.
  • the synthetic sequence SEQ ID NO: 11 (1979 bp) was digested with MluI and AgeI and connected to the MluI and AgeI sites of the 18BF007 plasmid, replacing the CMV-BGI-MCS-pA fragment, thereby constructing the 18BF008 plasmid.
  • the synthetic sequence SEQ ID NO: 12 (768bp) and sequence SEQ ID NO: 13 (765bp) were digested with ClaI and XhoI, respectively, and ligated to the ClaI and XhoI sites of the 18BF008 plasmid to construct 18BF085 and 18BF084 plasmids, respectively. .
  • the synthetic sequence SEQ ID NO: 10 (1496bp) was digested with SpeI and AgeI and connected to the AvrII and AgeI sites of the 18BF085 and 18BF084 plasmids, respectively, to construct the 19BF257 and 19BF256 plasmids, respectively.
  • Plasmid 19BF073 was digested with SpeI and AgeI, and the 3821bp fragment was recovered from the gel and ligated to the AvrII and AgeI sites of the 18BF085 and 18BF084 plasmids to construct the 19BF075 and 19BF074 plasmids, respectively.
  • plasmids 18BF019 and 18BF031 The synthetic sequence SEQ ID NO: 15 (1044bp) and sequence SEQ ID NO: 14 (1320bp) were digested with BamHI and XhoI and XhoI and BglII respectively and linked to the BamHI and BglII enzymes of the 18BF011 plasmid The site was cut to construct the 18BF019 plasmid.
  • the synthetic sequence SEQ ID NO: 16 (1806 bp) and sequence SEQ ID NO: 14 (1320 bp) were digested with BamHI and XhoI, and XhoI and BglII, respectively, and connected to the BamHI and BglII sites of the 18BF011 plasmid to construct the 18BF031 plasmid. .
  • Plasmids 18BF071 and 19BF254 were digested with Pad and AvrII respectively, and the DNA fragments were recovered by gels of 1762bp and 3838bp, respectively, and the two recovered fragments were connected to the 18BF004 plasmid. PacI and AvrII restriction sites were used to construct 18BF094 and 19BF255 plasmids, respectively.
  • Plasmid 18BF068 was digested with PacI, AvrII and PvuI, and the 2975bp DNA fragment was recovered from the gel, and the recovered DNA fragment was ligated to the PacI and AvrII restriction sites of the 18BF004 plasmid to construct the 18BF091 plasmid.
  • Plasmid 18BF074 was digested with PacI and PmeI, and a DNA fragment of 6428bp was recovered from the gel and ligated to the PacI and PmeI restriction sites of the 18BF004 plasmid to construct the 18BF096 plasmid.
  • Plasmid 19BF074 was digested with SpeI and PmeI, and a DNA fragment of 6513bp was recovered from the gel and ligated to the SpeI and PmeI sites of 18BF004 plasmid to construct 19BF252 plasmid.
  • plasmids 19BF081, 19BF217 and 19BF218 use pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252) as a template, hPGK-F (SEQ ID NO: 40) and hPGK-R (SEQ ID NO: 41) PCR amplification of PGK gene fragment (706bp) with primers; pGL3-Basic (Promega, E1751) as template, Luc-F (SEQ ID NO: 42) and Luc-R (SEQ ID NO: 43) as primers for PCR amplification luciferase gene fragment (1728bp), then use MluI and BamHI (538bp fragment recovered from gel) and BamHI and XhoI respectively to digest the two DNA fragments and connect them to the MluI and XhoI restriction sites of the 19BF126 plasmid to replace the original plasmid DNA fragment
  • MluI and BamHI 538bp fragment
  • TRIzol ThermoFisher 15596026
  • PBMC healthy human peripheral blood mononuclear cells
  • F8V1-F SEQ ID NO: 44
  • F8V1HA-R SEQ ID NO: 45
  • PCR product length 7116bp by DNA sequencing PCR product 18bp to 7070bp sequence is consistent with 172bp to 7224bp sequence in NM_000132.3 sequence
  • the 19BF215 plasmid was obtained.
  • BDDF8cHA (4434bp) is primer PCR to amplify two DNA fragments of 2317bp and 2154bp; fusion PCR method is used to connect these two DNA fragments and use F8V1F (SEQ ID NO:44) and F8V1HA-R (SEQ ID NO:45) as primers for PCR
  • the BDDF8cHA (4434bp) gene fragment was amplified; afterwards, it was digested with ClaI and XhoI and connected to the ClaI and XhoI restriction sites of the 19BF126 plasmid, replacing the original plasmid DNA fragment to construct the 19BF216 plasmid.
  • the synthetic sequence SEQ ID NO: 18 (3610 bp) was digested with SpeI and AgeI and connected to the SpeI and AgeI sites of the 18BF004 plasmid to construct the 19BF080 plasmid.
  • the synthetic sequence SEQ ID NO: 17 (1320 bp) was digested with XhoI and BglII and ligated to the XhoI and BamH I digestion sites of the 19BF080 plasmid to construct the 19BF214 plasmid.
  • Plasmids 18YYH26, 19BF215, and 19BF216 were digested with Pac I and Xho I, respectively, and the DNA fragments 2272 bp, 7792 bp, and 5110 bp were gel-recovered, respectively, and the recovered fragments were ligated to the Pac I and Xho I digestion sites of the 19BF214 plasmid, respectively.
  • the 19BF081, 19BF217 and 19BF218 plasmids were constructed respectively.
  • Plasmid 18BF022 was digested with Mlu I, Age I, and Pvu I, and a DNA fragment of 3052 bp was recovered from the gel and ligated to the Mlu I and Age I digestion sites of the 18BF004 plasmid to construct the 18BF033 plasmid.
  • the synthetic sequence SEQ ID NO: 18 (3610 bp) was digested with Spe I and Age I and connected to the Spe I and Age I sites of the 18BF007 plasmid, replacing the original plasmid DNA fragments to construct the 19BF077 plasmid.
  • Plasmid 19BF081 was digested with Pac I and Nde I, and a 3598 bp DNA fragment was recovered from the gel and ligated to the Pac I and Nde I digestion sites of the 19BF077 plasmid to construct the 19BF078 plasmid.
  • Example 2 Construction of lentivirus production cell lines using SB and PB double transposon systems
  • the specificity verification experiment was performed by transfecting the SB/PB transposon plasmid (18BF022/18BF033) and the SB/PB transposase plasmid (18BF019/18BF031) carrying EGFP into 293T cells, and then detecting the 293T cells in serial passages EGFP signal loss rate is used to evaluate whether the two sets of transposon systems can work cross-over.
  • the specific steps are as follows:
  • the transposon plasmids of SB and PB are 18BF022 and 18BF033, respectively ,
  • the reporter gene is EGFP.
  • the SB and PB transposase plasmids are 18BF019 and 18BF031, respectively.
  • the transposon plasmid and the transposase plasmid are co-transfected with calcium phosphate according to the molar mass of 10:1. The experiment has a total of 6 co-transfections.
  • SB transposon and SB transposase (18BF022+18BF019); (2) PB transposon and PB transposase (18BF033+18BF031); (3) SB transposon and PB transposase Transposase (18BF022+18BF031); (4) PB transposon and SB transposase (18BF033+18BF019); (5) SB transposon and empty plasmid (18BF022+18BF003); (6) PB transposon and Empty plasmid (18BF033+18BF003). After 6 hours of transfection, change the medium to a new complete DMEM medium.
  • Figure 2 shows the results of SB and PB specificity verification experiments.
  • the abscissa is the passage of cells, and the ordinate is the EGFP-positive ratio of each co-transfected cell combination (Figure 2A) and the median fluorescence intensity (MFI) ( Figure 2B) .
  • the positive ratio of EGFP in the P1 generation of the 6 groups of samples after transfection is between 61.1% and 79.1%, and the MFI is between 32685 and 44827.
  • the SB and PB transposon systems can efficiently integrate gene fragments in the cell genome without interfering with each other.
  • the genes rev, VSV-G, gag/pol used for packaging lentivirus and the virus carrying the target nucleic acid fragment are used in this example.
  • Genome transcription cassette and inducible expression system activator rtTA and/or repressor CymR protein coding sequence are constructed through different combinations, using SB and/or PB transposon system to transfect one or two times and carry out cell screening to construct stable production slow.
  • Figure 3 for the experimental process of the production cell line of the virus vector.
  • the combination described in this embodiment is only an example, and professionals in the industry can easily implement other combinations through the method shown in this embodiment.
  • the hPGK-Luciferase-IRES-EGFP sequence is used as the target nucleic acid fragment to develop and verify the method for constructing a stable lentivirus production cell line described in this disclosure.
  • This method is not affected by the specific target nucleic acid fragment in principle and can be any target nucleic acid. Fragment.
  • the first round of cell line transfection screening experiment 293T cells were seeded in a 60mm culture dish with 1.5E6 cells per culture dish and cultured for 24 hours.
  • the medium was 3ml DMEM complete medium, and the culture conditions were 37°C, 5% CO 2 .
  • Transfect cells according to the PEI method The operation method is as follows: During transfection, add 500 ⁇ L of transfection reagent per 60mm culture dish, which contains 9.5 ⁇ g of total plasmid amount. Refer to Table 5 for the amount of plasmid added in each experimental group.
  • the total amount of plasmid and PEI MAX( The mass ratio of Polysciences, 24765-1) is 1:4. The plasmid and PEI MAX are evenly mixed.
  • the cells were trypsinized and all the cells were seeded in 100 mm petri dishes (Corning, 430167).
  • Cell drug screening was carried out according to the screening resistance shown in Table 5, and continuously screened for at least 3 generations under the drug pressure until the cell line was stable, and the screening concentration of hygromycin (Hygromycin, Shenggong A600230-0001) was 200 ⁇ g/ml. After the cells grow stably, the drugs are removed and transferred to DMEM complete medium for culture.
  • Table 5 shows the cell line constructed in the first round of cell line transfection screening experiment, among which the genetically modified human embryonic kidney cell line EuLV-F2 was deposited on April 13, 2020
  • the serial number CGMCC No.19674 is deposited at the China Common Microbial Culture Collection Management Center (CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing).
  • the 8 cell lines of EuLV-F2, EuLV-F3, EuLV-F4, EuLV-F5, EuLV-F7, EuLV-F8, EuLV-F9 and EuLV-F10 were seeded in 8 60mm culture dishes with 1.5E6 cells respectively. Incubate for 24 hours.
  • the cells were transfected according to the PEI method, and the amount of plasmid added in each experimental group was referred to Table 6. After 24 hours of transfection, the cells were trypsinized and all inoculated in 100mm petri dishes. Perform cell drug screening according to the screening resistance shown in Table 6, and continue to screen for at least 3 generations under the drug pressure until the cell line is stable.
  • the screening concentration of puromycin (Puromycin, Aladdin P113126) is 2.5 ⁇ g/ml
  • Aladdin P113126 is 2.5 ⁇ g/ml
  • Aladdin B139600 Aladdin B139600 screening concentration
  • the second round of transfection screening experiment constructed EuLV-F2-S2, EuLV-F3-S3, EuLV-F4-S4, EuLV-F5-S5, EuLV-F7-S7, EuLV-F8-S8, EuLV-F9-S9
  • EuLV-F10-S10 There are 8 cell lines in total with EuLV-F10-S10.
  • the genetically modified human embryonic kidney cell line EuLV-F2-S2 cell line was deposited, and it was deposited at the China Common Microbial Culture Collection Management Center (CGMCC, Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing).
  • the HT1080 cell Luciferase virus titer detection method was used to detect the lentivirus-producing ability of the above-mentioned stable production cell line of lentivirus.
  • the 12 cell lines EuLV-NC-SB, EuLV-NC-PB, EuLV-F1, EuLV-F2-S2, EuLV-F3-S3, EuLV-F4-S4, EuLV-F5-S5 obtained after the screening are stabilized , EuLV-F6, EuLV-F7-S7, EuLV-F8-S8, EuLV-F9-S9, EuLV-F10-S10 were seeded in a 6-well plate (Corning 3516) according to 8E+05 cells per well and cultured at 37 Under the environment of °C and 5% CO 2 , the medium is DMEM complete medium.
  • DOX Doxycycline hydrochloride
  • DMEM complete medium with 5mmol/L sodium butyrate (Sigma, 303410) induces toxin production.
  • the method for detecting Luciferase virus titer of HT1080 cells is as follows: 24 hours after the above-mentioned experimental group and control group cells are added with the inducer, HT1080 cells are seeded in a 96-well plate (Corning 3916) with 1E4 cells per well, and the medium is DMEM. Complete medium. After 48 hours of induction of toxin production, the culture medium containing the lentivirus was collected and centrifuged at 14000 rpm for 10 minutes to collect the virus supernatant. One hour before adding the virus sample, the medium of HT1080 cells was replaced with a complete DMEM medium containing 8 ⁇ g/ml polybrene (Sigam, H9268).
  • Figure 4 shows the RLU value of the virus transfection titer measured by the HT1080 cell Luciferase virus titer detection method after the lentiviral stable production cells constructed according to different combinations of SB, PB transposon systems and lentiviral packaging genes are induced to produce toxins Test results.
  • the abscissa is the cell line constructed by different plasmid combinations, and the ordinate is the relative expression value of Luciferase RLU of the produced lentivirus after infection of HT1080 cells.
  • the results show that the use of different SB and/or PB transposons and different lentiviral packaging gene combinations can quickly construct a stable production cell line of lentivirus, and the stable cell lines constructed by various combinations can clearly detect the virus titer after induction.
  • a packaging cell containing an inducible expression system activator and/or repressor and lentiviral packaging genes rev, VSV-G, gag/pol is generally constructed first, and then based on this packaging cell.
  • the present disclosure preferably uses the SB transposon system to construct the Tet-ON induction system transactivator rtTA coding sequence and / Or Cumate induction system repressor CymR coding sequence and lentiviral packaging gene rev, VSV-G, gag-pol gene lentiviral vector packaging cell line (EuLV cells), and then use the PB transposon system to package the above lentiviral vector
  • the method of stably inserting the lentiviral genome transcription cassette carrying the target nucleic acid fragment into the cell line genome constructs a stable lentiviral production cell line.
  • Example 3 Optimization of induced expression of lentiviral genes rev, VSV-G and gag/pol
  • the main optimization conditions include three main aspects (1) the selection of the transactivator rtTA of the Tet-On induction system; (2) the optimization of the induction expression system includes the single regulation optimization of TRE adv and TRE 3G response elements based on the Tet-On induction system And TRE adv CuO and TRE 3G CuO response elements based on the Tet-On and Cumate composite inducible expression system; (3) and between the 3'end of the promoter or the inducible expression system response element and the 5'end of the regulated nucleic acid sequence Whether to connect a cleavable intron between.
  • this implementation design tested 8 combinations of induction response elements and introns and two Tet-On transactivators rtTA adv and rtTA 3G to regulate the lentiviral packaging genes gag/pol and VSV-G
  • the expression of and rev is optimized in terms of lentivirus production titer and leakage titer based on various conditions.
  • plasmid construction information refer to Example 1. The specific implementation steps are as follows:
  • Example 2 for the experimental procedures of 293T cell culture, PEI transfection, and hygromycin screening. Inoculate 293T cells in accordance with 1.5E+06 cells per 60mm culture dish, and culture them in DMEM (Sigam, D6429) complete medium supplemented with 10% FBS (ExCell, 11H116) at 37°C and 5% CO 2 . After culturing for 24 hours, the transfection was carried out according to the PEI method.
  • the total plasmid amount was 5.5 ⁇ g, and the transfection was carried out according to the molar ratio of plasmid 19BF074:18BF019 to 10:1 to obtain 293T-rtTA adv- CymR cells; according to the molar ratio of plasmid 19BF075:18BF019 to 10 :1 Transfection was performed to obtain 293T-rtTA 3G -CymR cells. After transfection, 200 ⁇ g/ml hygromycin was screened for at least three generations. After the cells grew under the pressure of the drug and the original 293T cells were the same, the following experiment was performed.
  • the cassette transfer vector plasmid, the virus was prepared by PEI transient transfection in the presence or absence of DOX and Cumate inducers, and the toxin production titer and leakage titer were evaluated by the HT1080 cell Luciferase virus titer detection method.
  • the eight rev test plasmids are 18BF072, 18BF071, 19BF249, 19BF248, 19BF247, 19BF246, 18BF070, and 18BF069;
  • the eight VSV-G test plasmids are 18BF068, 18BF067, 19BF245, 19BF244, 19BF243, 19BF242, 18BF066, and 18BF065;
  • 9 types of gag /pol test plasmids are 18BF074, 19BF131, 19BF130, 19BF251, 19BF250, 19BF129, 19BF128, 18BF076 and 19BF126;
  • the positive controls are plasmids pMD2.G, pRSV-Rev and pMDLg/pRRE.
  • the selected stable 293T-rtTA adv- CymR and 293T-rtTA 3G- CymR cells were seeded in a 24-well plate at 1.5E+05 cells per well, and the culture volume was 500 ⁇ l. After culturing for 24 hours, transfection was carried out according to the PEI method, and 50 ⁇ l of transfection mixture was added to each well, which contained 0.8 ⁇ g of total plasmid and 3.2 ⁇ g of PEI. In the total plasmid amount of 0.8 ⁇ g, the corresponding molar ratio of rev, VSV-G, gag/pol and 19BF081 plasmids is 1:1:1:1.
  • Figure 5 shows that the lentiviral packaging genes of rev ( Figure 5A), VSV-G ( Figure 5B) and gag/pol (Figure 5C) are induced to express differently in 293T-rtTA adv -CymR or 293T-rtTA 3G -CymR cells System response element regulation and Luciferase titer test results of induced toxin production titer and no induced leakage titer under the condition of whether a cleavable intron is connected between the 3'end of the response element and the 5'end of the regulated nucleic acid sequence .
  • the preferred regulatory elements for rev are TRE adv CuO (18BF070), TRE adv CuO-BGI (19BF249), TRE 3G (19BF246), TRE 3G CuO (19BF247) and TRE 3G CuO-BGI (18BF072), of which TRE 3G , TRE 3G CuO and TRE 3G CuO-BGI are preferred, and the corresponding average induced RLU and induced/leaked toxin production titer ratio are in 293T-rtTA adv- CymR and 293T- In rtTA 3G -CymR cells are (1) TRE 3G (19BF246): 6.80E+05 RLU, 135 times and 9.24E+05 RLU, 322 times; (2) TRE 3G CuO(19BF247): 6.34E+05 RLU , 275 times and 7.79E+05 RLU, 279 times
  • Tet-On single induction system further preferably transactivator is rtTA 3G , and the preferred response element is TRE 3G ; Tet-On and Cumate composite induction further preferably responds to TRE 3G , TRE 3G CuO and TRE 3G CuO-BGI.
  • VSV-G Based on the results of induced toxin production titer and no induced leakage titer, the preferred regulatory elements of VSV-G are TRE adv CuO (18BF066), TRE adv CuO-BGI (19BF245), TRE 3G -BGI (18BF067) and TRE 3G CuO-
  • the average value of induced RLU and the ratio of induced/leaked toxin production titers corresponding to BGI (18BF068) in 293T-rtTA adv -CymR and 293T-rtTA 3G -CymR cells are (1) TRE adv CuO (18BF066): 3.34E +05 RLU, 1323 times and 3.35E+05 RLU, 974 times; (2) TRE adv CuO-BGI (19BF245): 5.96E+05 RLU, 506 times and 4.62E+05 RLU, 886 times; (3) TRE 3G -BGI (18BF067):
  • the Tet-On single-induction system further preferably the transactivator is rtTA 3G , and the preferred response element is TRE 3G- BGI; the Tet-On and Cumate composite induction system further preferably responds to the element as TRE adv CuO-BGI, TRE 3G -BGI, TRE 3G CuO-BGI, more preferably TRE 3G CuO-BGI.
  • the detection titer Based on the results of induced toxin production titer and no induced leakage titer, under the condition that no intron (BGI) sequence is connected between the 3'end of the response element and the 5'end of the gag/pol coding sequence, whether it is an inducer or a non-intron In the induction group, the detection titer is below 1000 RLU, which is close to the background value; when the CMV promoter has no introns downstream (19BF126), the detection titer also drops to 19.4% of that with introns (18BF074), indicating The splicable intron sequence connected between the promoter and the gag/pol coding sequence is an important condition for the high expression of the gag/pol coding sequence.
  • BGI no intron
  • the average induced RLU and induced/leaked toxin production titer corresponding to the gag/pol plasmid containing introns in the transfection design are (1) TRE in 293T-rtTA adv -CymR and 293T-rtTA 3G -CymR cells, respectively adv -BGI (19BF250): 8.60E+05 RLU, 87 times and 6.43E+05 RLU, 135 times; (2) TRE adv CuO-BGI (19BF251): 9.01E+05 RLU, 413 times and 6.21E+05 RLU, 326 times; (3) TRE 3G -BGI (19BF130): 7.53E+05 RLU, 257 times and 7.16E+05 RLU, 333 times; and (4) TRE 3G CuO-BGI(19BF131): 8.22E+ 05 RLU, 641 times and 8.47E+0.5 RLU, 692 times; (5) CMV-BGI (18BF074): 1.
  • the regulatory elements of the gag/pol coding sequence are preferably CMV-BGI, TRE adv -BGI, TRE adv CuO-BGI, TRE 3G -BGI and TRE 3G CuO-BGI.
  • the preferred regulatory elements for Tet-On single induction are TRE 3G -BGI and CMV-BGI; the preferred response elements for Tet-On and Cumate induction are CMV-BGI, TRE adv -BGI, TRE adv CuO-BGI, TRE 3G -BGI and TRE 3G CuO-BGI, more preferably CMV-BGI, TRE adv CuO-BGI, TRE 3G CuO-BGI, and still more preferably CMV-BGI and TRE 3G CuO-BGI.
  • rev 18BF072, 19BF247 and 19BF246
  • VSV-G 18BF068 and 18BF067
  • gag /pol Plasmids 18BF074, 19BF131 and 19BF130 were combined in 293T-rtTA adv- CymR and 293T-rtTA 3G- CymR cells as shown in Table 7.
  • the preferred plasmid combination for Tet-On single induction is rev: 19BF246, VSV-G: 18BF067, and gag/pol: 18BF074 or 19BF130.
  • the corresponding average induced RLU and induced/leaked toxin production titer ratio in 293T-rtTA adv -CymR and 293T-rtTA 3G -CymR cells are 6.83E+05 RLU, 45 times and 7.28, respectively E+05 RLU, 167 times;
  • the corresponding average induced RLU and induced/leaked toxin production titer ratio in 293T-rtTA adv -CymR and 293T-rtTA 3G -CymR cells are 5.20E, respectively +05 RLU, 24 times and 6.94E+05 RLU, 176 times.
  • Tet-On and Cumate induce the optimal combination of plasmids and the average RLU of the three induction titers are from sample numbers 19BF246+18BF068+19BF131, 19BF246+18BF068+18BF074 and 19BF247+18BF068+18BF074 Plasmid combination.
  • the corresponding average induced RLU and induced/leaked toxin production titer ratio in 293T-rtTA adv -CymR and 293T-rtTA 3G -CymR cells are 1.28E+06 RLU, respectively , 1935 times and 1.46E+06RLU, 12032 times;
  • the corresponding average induced RLU and induced/leaked toxin production titer ratio are in 293T-rtTA adv- CymR and 293T-rtTA In 3G- CymR cells, 8.75E+05 RLU, 262 times and 1.07E+06 RLU, 975 times, respectively;
  • the preferred plasmid for rev is 19BF246 or 19BF247, more preferably 19BF246; the preferred plasmid for VSV-G is 18BF068; the preferred gag/pol plasmid is 19BF131 and 18BF074, and the preferred plasmid is 19BF131; trans
  • the activator is preferably rtTA 3G .
  • Example 4 Optimizing the ratio of rtTA-CymR, rev, VSV-G and gag/pol fragments stably integrated in the cell genome
  • High-efficiency production of lentivirus requires high viral gene expression after induction and an appropriate relative expression ratio of each viral gene.
  • the expression of each gene is mainly affected by the efficiency of the promoter or induction response element and the number of copies integrated in the cell genome.
  • the efficiency of the transposon system to stably integrate the target nucleic acid fragment in the cell genome is affected by the length of the gene fragment. The longer the target nucleic acid fragment, the lower the integration efficiency. Therefore, each transposon plasmid can be adjusted according to the length of the target nucleic acid fragment when constructing a stable cell line. The ratio during transient transfection is used to optimize the integration ratio of each fragment.
  • one of the group of preferred plasmid combinations optimized in Example 3, 19BF075, 19BF246, 18BF068 and 19BF131 plasmids is taken as an example.
  • the molar ratio is used to confirm the relationship between the amount of transfection plasmid added and the number of copies integrated in the host cell genome and the toxin-producing ability of the corresponding constructed cell; and then through the combination to optimize the molar ratio of single or multiple key genes in the cell line construction transient transfection confirmation Optimal genome integration copy number and molar ratio.
  • the optimal genomic integration ratio confirmed based on the plasmid selected in this example can be expressed according to the toxin production efficiency of each promoter/response element after induction shown in Example 3 or by comparison of other promoters/response elements not included in the present disclosure.
  • the efficiency derives the optimized insert copy number and molar ratio of other plasmid designs and combinations, so it is also in the protection scope of the claims supported by this example.
  • Example 2 for the experimental procedures of cell culture, PEI transfection, and hygromycin drug screening.
  • 293T cells were inoculated with 1.5E+06 cells per 60 mm culture dish and cultured in DMEM (Sigma, D6429) complete medium supplemented with 10% FBS (ExCell, 11H116) at 37° C. and 5% CO2. After culturing for 24 hours, transfection was carried out by PEI method and according to the content of each plasmid shown in Table 8 (the ratio of PEI to DNA was 4:1).
  • PEI and DNA mass ratio 4:1 by transient transfection of PEI. After 6 hours of transfection, change the medium and add inducer (final concentration of 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate and 5mmol/L sodium butyrate) to induce toxin production. After 48 hours, the culture supernatant was collected and the toxin production titers of EuLV-R1 to R17 cells were detected by the HT1080 cell Luciferase virus titer detection method.
  • inducer final concentration of 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate and 5mmol/L sodium butyrate
  • EuLV-R1 to R17 were seeded in a 24-well plate with 1.5E+05 cells per well. After culturing for 48 hours, the cells were collected to extract genomic DNA and use qPCR to absolutely quantify the integration of rtTA and CymR in the genome of EuLV-R1 to R5 cell lines Gene copy number; EuLV-R1 and EuLV-R6 to R9 cell line genome integrated rev gene copy number; EuLV-R1 and EuLV-R10 to R13 cell line genome integrated VSV-G gene copy number; EuLV-R1 And EuLV-R14 to R17 cell line genome integrated gag/pol gene copy number, the specific method is as follows.
  • Plasmids 19BF074, 18BF072, 18BF068 and 18BF074 were used as standard samples of HygroR (to detect rtTA and CymR copy numbers), rev, VSV-G and gag/pol genes, respectively, diluted with deionized water to 47.9ng/ ⁇ l, 23.8ng / ⁇ l, 30.0ng/ ⁇ l and 47.6ng/ ⁇ l, each plasmid at this concentration corresponds to 5.0E+09 copy number per ⁇ l sample.
  • the 4 standard samples were further diluted to 8.0E+06 copy number per microliter, and the sample was diluted in two-fold gradient to 1.56E+04 copy number per microliter, and the 10 diluted samples of the 4 standard products were made It is the qPCR standard curve of each gene to be tested.
  • the forward and reverse primers and probes of HygroR are: SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53; the forward and reverse primers and probes of rev are: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56; VSV-G forward , Reverse primers and probes are: SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; the forward and reverse primers and probes of gag/pol are: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50), and add water to 20 ⁇ l.
  • the curve and sample CT value calculate the copy number of the gene to be tested in each sample, and then calculate the copy number of the gene per cell based on the data obtained according to 6pg genomic DNA per cell.
  • the experimental results are shown in Figure 7.
  • the abscissa represents the molar ratio of transfected 4 plasmids when constructing cells; the bar graph corresponds to the left ordinate shows the RLU value of the induced toxin production of each cell; the line graph corresponds to the right ordinate shows each cell Integration of HygroR (detecting rtTA and CymR copy number), rev, VSV-G or gag/pol fragment copy number results in the genome.
  • HygroR detecting rtTA and CymR copy number
  • rev VSV-G or gag/pol fragment copy number results in the genome.
  • the results showed that (1) increasing the integrated copy number of rtTA 3G- CymR (HygroR probe experiment result) and rev fragment in the cell genome had no significant effect on the virus production ability of EuLV packaging cell line ( Figures 7A and 7B).
  • the toxin production titer increases with the increase of the gag/pol genome inserted copy number, and reaches the highest when it reaches more than 4.7 copies per cell. Toxin production titer 1.6E+06 RLU, continue to increase the number of inserted copies to more than 6 can not further improve the toxin production capacity (Figure 7D).
  • the integrated copy number and molar ratio of VSV-G and gag/pol fragments in the cell genome is the key to optimizing the toxin production capacity of EuLV packaging cell lines.
  • the total transfection volume of VSV-G and gag/pol fragments needs to be further optimized.
  • And molar ratio Consistent with the above method of constructing EuLV packaging cell line using SB transposon system, EuLV-VG1 to VG5 packaging cell lines were further constructed based on the contents of each plasmid shown in Table 9. After each cell line was selected to grow stably by hygromycin, it was the same as the above-mentioned 24-well plate lentivirus preparation method.
  • the 19BF081 plasmid was used as the target gene plasmid to prepare the virus and the luciferase virus titer was detected by HT1080 cells; at the same time, the qPCR quantitative method described above , To detect the integrated copy number of VSV-G and gag/pol fragments per cell in the genomic DNA of each cell line.
  • the experimental results are shown in Figure 8.
  • the results of the experiment are shown in Figure 8.
  • the abscissa represents the molar ratio of transfected 4 plasmids when constructing the cells; the bar graph corresponds to the left ordinate shows the RLU value of each cell's induced toxin production; the line graph corresponds to the right ordinate shows gag/
  • the results of the ratio of the number of integrated copies of pol and VSV-G fragments in the cell genome are as follows.
  • the gag/pol fragment has an integrated copy number of 2 to 8 copies per cell in the cell genome
  • the gag/pol and VSV-G fragment has an integrated copy number ratio in the cell genome between 1:1 and 4:1
  • the rtTA 3G- CymR and rev fragments can be integrated with at least one copy number in the cell genome to obtain a packaging cell line with high toxin production titer; further preferably, the gag/pol fragment has 4 to 6 integrated copies in the cell genome.
  • the number of copies per cell, the integration ratio of gag/pol and VSV-G fragments is between 2:1 to 3:1, rtTA 3G- CymR and rev fragments can get high production of toxic drops when at least one copy number is integrated in the cell genome Degree of packaging cell line.
  • Example 5 EuLV293T3rd lentiviral packaging cell line construction and high-throughput monoclonal screening
  • the EuLV293T3rd lentiviral packaging cell line construction process is shown in Figure 9, including three steps of plasmid transfection and cell selection, monoclonal selection and suspension adaptation. The experimental details and results are described in detail below.
  • the specific experimental method is as follows. Refer to Examples 2, 3 and 4 for the experimental procedures of cell culture, PEI transfection and hygromycin screening. Inoculate 293T (ATCC, CRL3216) cells in a 60mm culture dish with 1.5E+06 cells per culture dish. The medium is 3ml DMEM (Sigma, D6429) supplemented with 10% FBS (ExCell, 11H116). Incubate for 24 hours at 37°C under 5% CO 2 conditions.
  • Plasmid transfection was carried out by PEI transfection method, the total plasmid amount was 5 ⁇ g, the mass ratio of PEI to total plasmid was 4:1, and the molar ratio of plasmid 19BF257:19BF246:18BF067:19BF130:18BF019 was 3:3:2:12: 2 Transfection was carried out to construct EuLV293T3rd-SB16 cells; according to the plasmid 19BF075:19BF246:18BF068:19BF131:18BF019 molar ratio of 3:3:2:12:2, EuLV293T3rd-SB28 was constructed by transfection.
  • the specific experimental method is as follows.
  • the screening process of SB16 and SB28 high-yielding monoclonal cell lines is shown in Figure 9.
  • SB16 and SB28 cells were inoculated into 10 384-well plates with one cell per well by the limiting dilution method for the first round of screening.
  • the screening basis was as follows: ( 1) There is one and only one monoclonal cell; (2) The cell morphology is normal; (3) The cell growth rate is the same as that of the original 293T cell.
  • the HT1080 cell Luciferase virus titer detection method was used to detect the induced toxin production titer of each monoclonal cell when the cells were expanded to a 96-well plate, a 24-well plate, and a 6-well plate culture system.
  • Figure 10 shows the distribution of the ratio of monoclonal cell toxin production to the standard product at each screening stage.
  • the HT1080 cell Luciferase virus detection method to detect the monoclonal cell toxin production titer RLU value and count the number of monoclonal cells in each interval from 0 to 100,000 according to the interval of 500.
  • the abscissa is the upper limit and standard of each interval.
  • the ratio of the RLU value of the product detection (expressed as the logarithm of the base 2), and the ordinate is the number of monoclonal cells in each statistical range.
  • Figure 10A shows the screening results of the SB16 cell line.
  • FIG. 10B is the screening result of the SB28 cell line, respectively in 96 wells. 87, 30, and 9 monoclonal cells were selected in the screening steps of plate, 24-well plate and 6-well plate.
  • the specific operation method of cell screening is as follows.
  • 384-well plate monoclonal screening the specific experimental steps are as follows.
  • the SB16 and SB28 initial cells constructed above were cultured in DMEM complete medium at 37°C and 5% CO 2 to a monolayer of 70% confluence.
  • the cells were seeded in a 384-well plate at a density of one cell per well, and 5 384-well plates were seeded with SB16 and SB28 cells.
  • an inverted microscope Olympus IX71
  • the monoclonal cells with a confluence greater than 50% were seeded into a 96-well plate at 2E+04 cells per well, and a total of SB16 monoclonal cells were obtained.
  • the specific experimental procedures for monoclonal screening in 96-well plates are as follows. After culturing the cells obtained from the monoclonal screening step of the 384-well plate for 24 hours, inoculate 5E+03 and 2.5E+04 cells per well in two new 96-well plates for subculture and lentivirus induction respectively. Detection. In the step of induction of toxin production by monoclonal cells, a positive control of lentivirus was prepared by transiently transfecting 293T cells. The method is as follows: 293T cells were seeded into a 96-well plate at 2.5E+04 cells per well, and cultured for 24 hours.
  • the virus was prepared by transiently transfecting plasmids by the PEI method (total DNA amount is 0.3 ⁇ g, wherein the molar ratio of plasmid 19BF081:pMD2.G:pMDLg/PRRE:pRSV-Rev is 1:1:1:1, total DNA and PEIMAX The mass ratio is 1:4). Two hours after transfection, an inducer (final concentration of 2mmol/L sodium butyrate, 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate) was added, and after culturing for 48 hours, supernatant virus was collected by centrifugation at 4500rpm for 15 minutes.
  • an inducer final concentration of 2mmol/L sodium butyrate, 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate
  • the method for inducing toxin production by monoclonal cells in 96-well plates is as follows: the above-mentioned monoclonal cells to be tested inoculated into 96-well plates at 2.5E+04 cells per well are cultured for 24 hours, and transfection reagents are added (total DNA amount is 0.3 ⁇ g, the addition amount of 19BF081 is the same as the above positive control, the remaining plasmid amount is made up to 0.3 ⁇ g with 18BF003, and the mass ratio of total DNA to PEI MAX is 1:4). Afterwards, the method for inducing toxin production and recovering the virus supernatant is the same as the method for preparing the above-mentioned positive control.
  • the RLU value of the positive control and each monoclonal cell-induced toxin production sample was detected.
  • the monoclonal cells with RLU values higher than the positive control were selected from high to low, and the SB16 monoclonal cell 87 strain and the SB28 cell monoclonal 94 strain were obtained.
  • the high-yield monoclonal cell screening process of 24-well plate and 6-well plate is basically the same as the above-mentioned 96-well plate monoclonal screening.
  • the seeding density of 24-well plate was 2E+04 cells per well, and that of 6-well plate was 2E+05 cells.
  • the seeding density of the 24-well plate was 1E+05 cells per well, and the seeding density of the 6-well plate was 8E+05 cells.
  • the total amount of DNA per well in a 24-well plate is 1.2 ⁇ g and that in a 6-well plate is 5 ⁇ g.
  • Other transfection conditions such as the ratio of each plasmid and the ratio of DNA to PEI are consistent with the above experimental method.
  • the HT1080 cell Luciferase virus titer detection method in the 24-well plate monoclonal screening stage, 30 high-yielding packaging cell lines were further screened from 87 strains of SB16 and 94 strains of SB28 monoclonal cells. In the monoclonal screening stage of the 6-well plate, 9 SB16 and SB28 monoclonal high-yielding packaging cells were further screened from each of the 30 cells. The final screening monoclonal cell numbers are shown in Table 11.
  • the specific experimental method is as follows.
  • the 18 monoclonal cells obtained in Table 11 were inoculated into a 125ml shake flask (Corning 431143) at 5E+05 cells/ml, 20ml per bottle for suspension adaptation, and the medium was Freestyle293 ( FreeStyle TM 293Expression Medium 12338018), the culture condition is 37°C, 5% CO 2 , 1.9 cm shake distance shaker, rotating speed 140 rpm.
  • the cells were grown to 2.5E+06 cells/ml, they were subcultured and cultured continuously until there was no obvious cell clumping and the doubling time was less than 24 hours to obtain a suspension culture cell line of the above-mentioned 18 cell lines.
  • the virus preparation method of the suspension cultured SB16 and SB28 cell lines is as follows. After the above cells were cultured in suspension to a density of 2.5E+06 cells/ml, the cells were harvested by centrifugation at 1000 rpm for 3 minutes, resuspended in fresh Freestyle293 medium and adjusted to a cell density of 4E+06 cells/ml. Transfer the cell suspension 0.5ml per well to a 96 deep well plate, and add 50 ⁇ L PEI transfection reagent prepared in DMEM medium, which contains 7.5 ⁇ g PEI MAX and 2.5 ⁇ g total DNA (which contains 1 ⁇ g 18BF081 and 1.5 ⁇ g 18BF003 ). After mixing, continue culturing on a culture shaker.
  • the culture conditions are 37°C, 5% CO2, 1.0cm shaking distance shaker, and rotating speed 1000rpm.
  • an inducer final concentration of 2mmol/L sodium butyrate, 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate
  • the virus titer was detected according to the HT1080 cell Luciferase virus titer detection method described in Example 2. The detection result is shown in Figure 11, the abscissa is the monoclonal number, and the ordinate is the titer detection result, expressed in RLU.
  • the results show that all monoclonal cells can adapt to serum-free suspension culture conditions.
  • the titers of different monoclonal cells transiently transfected with 19BF081 plasmid under suspension culture conditions are between 9.96E+04 to 4.68E+06 RLU, and the average titre is between 9.96E+04 and 4.68E+06 RLU.
  • the degree is 8.56E+05 RLU, and the median value is 5.7E+05 RLU.
  • Example 6 Detection of optimization degree of EuLV293T3rd optimal toxin-producing packaging cell line
  • the optimal toxin-producing monoclonal packaging cell line selected in Example 5 was transfected with other rev, gag/pol, and VSV regulated by a constitutively active promoter while packaging the lentivirus by transient transfection and transferring the vector plasmid.
  • -G lentiviral packaging gene expression plasmid to detect whether the continued increase in the expression of viral packaging genes can further increase the toxin production titer of packaging cells, so as to determine whether each packaging gene fragment in the cell line has reached saturation and the relative proportion of each virus packaging gene fragment Whether to achieve the optimization. From Example 5, 6 high-yielding adherent cell lines and 5 high-yielding suspension cell lines were selected for the above experiment. The cell line names and numbers are shown in Table 12.
  • Adherent culture high-yield cell line lentivirus packaging gene supplement experiment The adherent high-yield cell line shown in Table 12 was cultured at 37°C and 5% CO 2 with a complete DMEM medium. After that, each type of cell was seeded into different wells of a 6-well plate according to 8E+05 cells per well, and transfection experiments were performed according to the calcium phosphate transfection method in Example 2. During transfection, add 200 ⁇ L transfection reagent to each well, containing 0.12mol/L calcium chloride, 1xHEPES buffer, and 6.8 ⁇ g total plasmid amount. Refer to Table 13 for the combination and content of plasmids in each experimental group.
  • the 18BF084 plasmid expresses the rtTA adv transactivator; 19BF081 is the transfer plasmid transcribing the lentiviral genome carrying the hPGK-Luciferase-IRES-EGFP target nucleic acid fragment; pMD2.G (Addgene, 12259) Express VSV-G protein; pMDLg/pRRE express gag and pol protein; pRSV-Rev express rev protein. After 6 hours of transfection, change the medium to DMEM complete medium containing 5mM sodium butyrate and add 1 ⁇ g/ml DOX and 200 ⁇ g/ml Cumate inducer to the samples that need to be added with inducer according to Table 13.
  • the HT1080 cell Luciferase virus titer detection method described in Example 2 was used to detect the lentivirus-producing ability of each adherent cell line, and the results are shown in FIG. 12.
  • the abscissa is the plasmid transfection combination of each experimental group, and the ordinate is the RLU value of Luciferase expression in the corresponding experimental group infected HT1080 cells.
  • the dotted line in the figure indicates the RLU value of the titre result of the adherent cell line only transiently translating the 19BF081 transfer plasmid to induce toxin production under this condition, and the solid line in the figure is the positive control result of 293T.
  • the transfection titers of the 6 packaging cells were between 9.9E+05 RLU and 5.8E+06 RLU when the lentivirus packaging genes were not supplemented.
  • lentiviral packaging genes such as rev, VSV-G, and gag/pol
  • most of the high-yielding cell lines did not increase significantly.
  • the cells with a greater increase in toxin production are cells with a lower initial toxin production titer.
  • rtTA, rev, VSV-G, gag/pol single gene or multiple genes most cell lines have reduced toxin production titers.
  • gag/pol insert copy number and expression level have an effect on the cell line toxin production titer. The importance of.
  • Suspension culture high-yield cell line packaging gene supplement experiment The suspension cell lines shown in Table 12 were cultured at 37°C, 8% CO 2 , the shaking distance was 1.9 cm, and the number of revolutions was 140 rpm.
  • the medium was FreeStyle ( FreeStyle TM 293 Expression Medium, 12338018).
  • each suspension cell line was seeded into a 96-well deep-well plate with 4E+06 cells per well, with a culture volume of 1 ml.
  • the cells were transfected according to the PEI method in Example 5. 100 ⁇ L of transfection sample was added to each well during transfection, containing 2.5 ⁇ g of total plasmid and 7.5 ⁇ g of PEI.
  • the abscissa is the plasmid transfection combination of each experimental group, and the ordinate is the RLU value of Luciferase expression in the corresponding experimental group infected HT1080 cells.
  • the dotted line in the figure indicates the RLU value of the titre result of the suspension cell line only transiently transfecting the 19BF081 transfer plasmid to induce toxin production under this condition, and the solid line in the figure is the positive control result of 293T.
  • the results showed that the RLU results of the 5 suspension cells without supplementing the lentivirus packaging gene were between 8.9E+05 RLU and 4.0E+06 RLU.
  • the toxin production titer of most high-yielding cells did not increase significantly.
  • the cells with a greater increase in toxin production are cells with a lower initial toxin production titer.
  • rtTA, rev, VSV-G, gag/pol single gene or multiple genes most cell lines have different levels of toxin production titers. Only some cells benefit from further supplementation of gag/pol genes, indicating that most screening The ratio of packaging genes of the obtained cell lines has been fully optimized, and further supplementation of virus packaging genes will reduce the cell line toxin production titer.
  • Table 14 Names and dosages of plasmids in each experimental group in the supplementation experiment of lentivirus packaging genes of high-yielding cell lines in suspension culture
  • Example 7 Construction of multiple lentivirus production cell lines based on high-yield packaging cell lines EuLV293T3rd-SB16-3D3 and EuLV293T3rd-SB28-1C2
  • the transfer vector plasmids containing three different target nucleic acid fragments were stably integrated into the genomes of EuLV293T3rd-SB16-3D3 (hereinafter referred to as 3D3) and EuLV293T3rd-SB28-1C2 (hereinafter referred to as 1C2) packaging cells through the PB transposon system.
  • 3D3 EuLV293T3rd-SB16-3D3
  • 1C2 EuLV293T3rd-SB28-1C2
  • the transfer vector plasmids of the above three viral genome transcription cassettes carrying the target nucleic acid fragments are: (1) 19BF081 (hPGK-Luciferase-IRES-EGFP, lentiviral genome length 5.7 kbp); (2) 19BF218 (deleting B protein domain) The coagulation factor 8 sequence, CMV-BDDF8cHA-IRES-EGFP, the lentiviral genome length is 8.5 kbp); (3) 19BF217 (the coagulation factor 8 full-length sequence, CMV-F8cHA-IRES-EGFP, the lentiviral genome length is 11.2 kbp).
  • the above-mentioned target nucleic acid fragments are all connected with the IRES-EGFP sequence.
  • the toxin-producing ability of these stable lentivirus production cell lines was compared with the method of producing lentivirus by transient transfection of 293T under the conditions of adherent culture and the same transfer plasmid.
  • a production cell line for the stable production of 3 different lentiviruses was constructed.
  • the specific experimental methods are as follows. Inoculate 3D3 and 1C2 cells in a 6-well plate with 1.5E+06 cells per well. The medium is 3ml DMEM complete medium. After culturing for 24 hours at 37°C under 5% CO 2 conditions, transfection is carried out according to the PEI method. The total amount of plasmid was 5.5 ⁇ g, of which the transfection was carried out according to the molar ratio of the transfer plasmid and the PB transposase plasmid (18BF031) of 10:1, and the total amount of PEI was 22 ⁇ g.
  • the transfer plasmids were 19BF081, 19BF218 and 19BF217. 24 hours after transfection, 2.5 ⁇ g/ml puromycin (Puromycin, Aladdin P113126) was added to screen for at least 3 passages until the cells were stable. A total of 6 stable lentivirus production cell lines were obtained, 3D3-19BF081, 3D3-19BF218, 3D3 -19BF217, 1C2-19BF081, 1C2-19BF218 and 1C2-19BF217.
  • the toxin production method of the stable lentivirus production cell line is compared with that of the transient transfection method.
  • the specific experimental method is as follows. Inoculate the 6 kinds of cells obtained by the above screening in each well of 8E+05 cells in a 6-well plate.
  • the medium is DMEM complete medium. After culturing for 24 hours at 37°C under 5% CO 2 condition, replace with new DMEM complete medium. And add inducer (final concentration of 2mmol/L sodium butyrate, 1 ⁇ g/ml DOX and 200 ⁇ g/ml Cumate). After culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes.
  • the method of preparing the virus by transiently transfecting 293T cells is the same as the method of preparing the positive control lentivirus described in Example 5.
  • the molar ratio of the transfer vector plasmid: pMD2.G: pMDLg/PRRE: pRSV-Rev is 1:1:1:1 ,
  • the transfer vector plasmids are 19BF081, 19BF218 and 19BF217 respectively.
  • HT1080 cells ATCC CCL-121 were seeded in a 24-well plate (Corning 3524) with 5E+04 cells per well under the same culture conditions as 293T. After 24 hours, take at least 3 wells and digest them with trypsin and count them. Replace the other wells with 500 ⁇ L of DMEM complete medium containing 8 ⁇ g/mL polybrene (Sigma H9268). Dilute the virus solution with DMEM complete medium to multiple detection concentrations, and then add 100 ⁇ L of diluted virus solution to the 24-well plate for culturing HT1080 cells.
  • the titers of the three 3D3 stable production cell lines carrying 19BF081 (5.7kb), 19BF217 (11.2kb) and 19BF218 (8.5kb) transfer vectors were 1.11E6TU(EGFP)/ml and 2.33E5TU(EGFP), respectively /ml and 1.05E6TU(EGFP)/ml, which are 6.14, 8.27 and 5.01 times of the positive control of transient transfection, respectively;
  • the toxin production titers of the three stable 1C2 lentivirus production cell lines carrying the above three transfer vectors are 1.59, respectively E6TU(EGFP)/ml, 3.55E5TU(EGFP)/ml and 1.81E6TU(EGFP)/ml are 8.81, 12.6 and 8.62 times of the positive control of transient transfection, respectively.
  • stable lentivirus production cell lines can produce toxin titers more than one order of magnitude higher when expressing long-term nucleic acid fragments than transient methods, and have a wider range of industry applications, such as constructing expression when using gene therapy methods to treat hemophilia
  • a full-length coagulation factor 8 lentiviral vector (take 19BF217 as an example).
  • the toxin production titers and leaked virus titers of 3D3-19BF081 and 1C2-19BF081 cells were tested under suspension culture conditions.
  • the specific experimental methods are as follows. According to the cell suspension adaptation method described in Example 5, 3D3-19BF081 and 1C2-19BF081 cells were adapted to suspension culture. When the suspended cells grow to 2.5E+06 cells/ml, harvest the cells by centrifugation at 1000 rp/min for 3 minutes, and adjust the cell density to 4E+06 cells/ml with fresh Freestyle293 medium.
  • the cell suspension to a 50ml centrifuge tube according to 5ml per tube, set the induction group with the addition of inducer (final concentration of 2mmol/L sodium butyrate, 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate) and no DMEM medium.
  • inducer final concentration of 2mmol/L sodium butyrate, 1 ⁇ g/ml DOX, 200 ⁇ g/ml Cumate
  • the induction group was then placed on a cell culture shaker to continue culturing for 48 hours, and then centrifuged at 4500 rpm for 15 minutes to collect the supernatant virus.
  • the positive control is a virus prepared by transiently transfecting 293T cells cultured in the same suspension culture condition. The specific method is as follows.
  • Plasmid transfection was carried out by PEI transfection method, the total plasmid amount was 12.5 ⁇ g, the mass ratio of PEI to total plasmid was 4:1, and the molar ratio of plasmid 19BF081:pMD2.G:pMDLg/PRRE 1:pRSV-Rev was 1: 1:1:1.
  • sodium butyrate was added at a final concentration of 2mmol/L, and the culture was continued until the virus sample was recovered by centrifugation. Afterwards, the RLU value of each sample was detected according to the HT1080 cell Luciferase virus titer detection method described in Example 2, and the results are shown in FIG. 15.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了用于生产携带目的核酸片段的逆转录病毒载体或慢病毒载体的生产细胞及制备方法，包括利用睡美人转座子系统和PiggyBac转座子系统将各基因的序列整合在宿主细胞的基因组中。还提供了用于逆转录病毒载体的目的核酸片段和/或外包膜蛋白替换系统、方法和生产细胞。还提供了携带目的核酸片段的逆转录病毒载体及其在制备用于向细胞递送目的核酸片段的试剂中的用途。还提供了制备用于生产慢病毒载体的包装/生产细胞及其制备方法和生产慢病毒载体的方法，其中gag、pol和rev基因以及VSV-G的编码序列中的一种或多种的转录在Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下。

Description

用于逆转录病毒载体的生产细胞和包装细胞及其制备方法 技术领域
本公开涉及病毒载体领域，具体地，涉及逆转录病毒尤其是慢病毒载体领域；更具体地，涉及逆转录病毒尤其是慢病毒载体的制备方法，用于逆转录病毒尤其是慢病毒载体制备的生产/包装细胞以及所述细胞的制备方法。
背景技术
逆转录病毒，又称反转录病毒，属于RNA病毒中的一类，是具有包膜的双链RNA病毒，其主要特征在于能够将其基因组由RNA“逆转录”为DNA。病毒粒的直径一般为100nm左右，并且包含与核衣壳(NC)蛋白形成复合体的二聚基因组(两条相同的单股正链RNA)。其基因组被包封在蛋白衣壳(CA)中，该衣壳还包含具有酶活性的蛋白，即反转录蛋白酶(RT)，整合酶(IN)和蛋白酶(PR)，这些酶是病毒感染所必需的。基质蛋白(MA)在衣壳核外形成一个层，该层与包膜相互作用，包膜是来源于宿主细胞膜并且包围病毒核心颗粒的脂质双层。锚定在该包膜上的是病毒包膜糖蛋白(Env)，其负责识别宿主细胞上的特异性受体并且启动感染过程。包膜蛋白由两个亚基形成，其分别是将所述蛋白锚定在脂膜中的跨膜(TM)亚基以及与细胞受体结合的表面(SU)亚基。
γ-逆转录病毒载体，是最通常被使用的逆转录病毒载体，在2017年所有申报基因治疗临床实验所用的转染方法中占17.3％。目前，人们对于来源于复杂的逆转录病毒如人免疫缺陷病毒(HIV-1)的慢病毒(Lentiviral vector)的兴趣与日俱增，从2012年的2.9％基因治疗临床试验占比上升到2017年的7.3％。这是因为慢病毒能够转导非分裂的细胞，该特性使其区别于其他病毒载体(包括γ-逆转录病毒载体)。另外，慢病毒相对于逆转录病毒有更有利的基因插入位点谱系。逆转录病毒和慢病毒载体最为吸引人的特性在于：作为基因递送工具，其基因递送能力可达9kb；较小的患者免疫反应和较好的经临床证实的安全性；在体内和体外的高转导效率；以及能够永久性地将外源基因整合到靶细胞基因组，使递送的基因具有长效的表达。
原型慢病毒载体系统是基于HIV-1病毒开发的，HIV-1是一种已被充分研究的人病原病毒。除了HIV-1，其他慢病毒也已被开发用作基因递送载体(TV)，但大多未达到临床研究阶段，如HIV-2，猿免疫缺陷病毒(Simian immunodeficiency virus,SIV)，或者非灵长类动物慢病毒，包括猫免疫缺陷病毒(Feline immunodeficiency virus,FIV)，牛免疫缺陷病毒(Bovine immunodeficiency virus,BIV)或山羊关节炎-脑炎病毒(Caprine arthritis-encephalitis virus，CAEV)。仅基于马传染性贫血病毒(Equine infectious anemia virus，EIAV)的载体已被开发至治疗帕金森的临床使用阶段(ProSavin)。
主要出于对HIV-1在人体中具有致病性所带来的安全性问题的考虑，目前开发有三代慢病毒载体系统。第一代慢病毒载体系统是以三质粒系统为代表，该系统由包装质粒、包膜质粒及载有携带目的核酸 片段的病毒基因组转录盒(transcriptional casstte)的转移载体(transfer vector)质粒3种质粒组成。包装质粒源自HIV-1前病毒基因组，其5’端LTR由巨细胞病毒早期启动子取代，3’LTR由猿猴病毒-40聚腺苷酸(SV40 polyA)序列取代，并且删除了HIV-1的包膜基因env。此包装质粒同时表达rev、vif、vpr、vpu和nef辅助基因。被删除的HIV-1包膜基因被替换为水疱性口炎病毒(Vesicular stomatitis virus)的包膜蛋白基因VSV-G并由包膜质粒表达。载有携带目的核酸片段的病毒基因组转录盒的转移载体质粒携带了HIV-1的5’端LTR，和全部5’端非翻译区域，300bp左右的5’端gag基因，中心多嘌呤束(cppt)片段，另外还带有rev应答元件(RRE)片段。此转移载体质粒用于克隆目的核酸片段并在病毒组装时提供病毒基因组RNA。第二代慢病毒载体系统是在第一代的基础上进行改进得到的，其在包装质粒中删除了HIV-1的所有辅助基因(vif、vpr、vpu和nef基因)。这些辅助基因的去除并不影响病毒的滴度和感染能力，同时增加了载体的安全性。第三代慢病毒载体系统由四质粒组成，其将rev基因从包装质粒上移除并单独放在另一个包装质粒上。此外，第三代慢病毒载体系统同时增加了两个安全特性：第一个安全特性是构建自身失活的慢病毒转移载体质粒，即删除了病毒基因组转录盒中3′LTR的U3区，使慢病毒载体在完成逆转录反应后永久性的失去5’LTR和3’LTR的U3区增强子及启动子片段，使得即使在此时存在所有的病毒蛋白也因为不能再转录出病毒的基因组RNA而不能成功包装病毒，因此第三代转移载体质粒也被称为自我失活(self-inactivating,SIN)转移载体质粒。同时删除了U3区也大大降低了载体基因插入宿主细胞的致癌性。第二个安全特性是去除了有转录反式激活功能的tat基因，用异源启动子序列代替5’LTR的U3区增强子及启动子序列转录病毒基因组RNA，并且在转录慢病毒基因组RNA时，异源启动子自身不能转录，因此进一步保证慢病毒载体仅能包装转染一次。第三代慢病毒系统仅保留了原始的HIV-1基因组中的gag、pol和rev基因序列，因此第三代慢病毒载体系统更加安全。
逆转录病毒/慢病毒载体可以通过替换不同的异源包膜糖蛋白而具有不同的假型(pseudotype)，比如替换为慢病毒(Lentivirus，如人、猿、猫、牛免疫缺陷病毒(immunodeficiency virus)、山羊关节炎-脑炎病毒(caprine arthritis-encephalitis virus)、马传染性贫血病毒(Equine infectious anemia virus)等)包膜蛋白、逆转录病毒(Retroviruse,如鼠白血病病毒(Murine leukemia virus,10A1,4070A)、长臂猿猿白血病病毒(Gibbon ape leukemia virus)、猫白血病病毒(Feline leukemia virus,RD114)、两性逆转录病毒(Amphotropic retrovirus)、嗜性逆转录病毒(Ecotropic retrovirus)、狒狒猿白血病病毒(Baboon ape leukemia virus)等)包膜蛋白、副粘病毒(Paramyxoviruses,如麻疹病毒(Measles virus)、尼帕病毒(Nipah virus)等)包膜蛋白、弹状病毒(Rhabdoviruses,如狂犬病病毒(Rabies virus)、莫科拉病毒(Mokola virus)等)包膜蛋白、丝状病毒(Filoviruses,如埃博拉病毒扎伊尔病毒(Ebola Zaire virus)等)包膜蛋白、沙眼病毒(Arenaviruses，如淋巴细胞脉络膜脑膜炎病毒(Lymphocytic choriomeningitis virus)等)包膜蛋白、杆状病毒(Baculovirus)包膜蛋白、甲病毒(Alphaviruses，如基孔肯雅病毒(Chikungunya virus)、罗斯河病毒(Ross River virus)、塞姆利基森林病毒(Semliki Forest virus)、信德比斯病毒(Sindbis virus)、委内瑞拉马 脑炎病毒(Venezuelan equine encephalitis virus)、西部马脑炎病毒(Western equine encephalitis virus)等)包膜蛋白、正粘病毒(Orthomyxoviruses,如流感病毒(Influenza virus)、禽瘟病毒(Fowl Plague Virus)等)包膜蛋白、疱疹病毒(Vesiculoviruses,如水泡性口炎病毒(Vesicular stomatitis virus)、昌迪普拉病毒和皮里病毒(Chandipura virus and Piry virus)等)包膜蛋白，目前大多数的慢病毒载体制备使用的都是水疱性口炎病毒包膜糖蛋白(VSV-G)，这是由于此种糖蛋白使得慢病毒载体具有大范围的转导谱(transduction spectrum)以及在下游处理过程中的更好的稳定性。
目前大部分逆转录病毒/慢病毒载体优选使用哺乳动物细胞系做为宿主细胞制备，最广泛使用的是293T或HEK293细胞系以及基于它们筛选或改造的衍生细胞系。HEK293细胞是转染腺病毒E1A基因的人肾上皮细胞系，293T细胞由HEK293细胞派生，同时表达SV40大T抗原，含有SV40复制起始点与启动子区的质粒可以在293T细胞内复制并在一段时间内维持高质粒拷贝数，提高质粒携带基因的蛋白表达量。此外，与HEK293细胞相比，293T细胞具有更快的细胞生长速度和更高的转染效率。以上293T细胞特点使得其具有更高的病毒载体制备效率。另外，根据文献报道，可用于病毒载体的生产/包装细胞还包括哺乳动物细胞HepG2细胞、CHO细胞、BHK细胞、COS细胞、NIH/3T3细胞、Vero细胞、HT1080细胞、Te671细胞、CEM细胞、NSO细胞和PerC6细胞。
目前的逆转录病毒/慢病毒载体的制备方法一般为在哺乳动物宿主细胞内，通常是来源于鼠或人的细胞内，分别转入gag基因、pol基因、rev基因(用于慢病毒载体)、包膜糖蛋白基因，以及携带目的核酸片段的病毒基因组转录盒(包含转录包装进逆转录病毒/慢病毒的RNA基因组的启动子；逆转录病毒/慢病毒基因组包装及转染所需的各种顺式作用序列，如5’LTR、PBS、ψ包装信号、cppt(用于慢病毒载体)、RRE(用于慢病毒载体)、ppt和3’LTR序列等；待转导的目的核酸片段，3’端的聚腺苷酸信号等)。这可以通过将含上述基因的构建体比如质粒，瞬时转染到上述宿主细胞中并且继之以24-72小时的病毒载体生产和收获实现(瞬时生产)，也可以通过在上述宿主细胞基因组中稳定整合上述基因形成稳定生产细胞系(producer cell line)以进行持续生产来实现(稳定生产)。
瞬时生产利用转染质粒方法来引入病毒基因构建体，所述方法使用可以与带负电的DNA形成复合物的阳离子试剂，从而允许其经由内吞作用被细胞摄入。聚乙烯亚胺(PEI)是最被广为使用且效率最高的阳离子试剂之一，目前在临床以及工业上大都利用PEI转染法来引入上述构建体，但其在工艺放大后存在工艺不稳定、产毒滴度低等主要问题。也可以使用其他方法，如磷酸钙沉淀、阳离子脂质体络合和非脂质体转染试剂，比如Lipofectamine和
Figure PCTCN2020115522-appb-000001
然而，这些方法仅可用于小规模生产或仅用于研究目的，因为其要么难以放大规模要么太过昂贵。备选地，病毒感染也已被开发和验证用于逆转录病毒/慢病毒载体生产，其使用杆状病毒或腺病毒来引入慢病毒基因构建体。然而，此种方法需要在下游额外地对逆转录病毒/慢病毒载体和杆状病毒或腺病毒进行分离以符合临床级的病毒生产标准，并且相比于质粒DNA转染法，使用杆状病毒或腺病毒来制备逆转录病毒/慢病毒载体的工艺复杂而最终转染滴度不高。 备选地，连续流电穿孔技术也已经被开发用于大体积细胞转染，原理上也可以被用于逆转录病毒/慢病毒的瞬时转染生产，但此技术依然受限于工艺放大能力以及昂贵的设备和耗材。
构建逆转录病毒/慢病毒稳定生产细胞系依赖于将病毒基因构建体分别整合到细胞基因组中以允许其进行组成型表达或被调控表达。通常，首先将gag、pol、rev(用于慢病毒载体)和env基因同时或顺序转入细胞并通过相应的抗性筛选和克隆选择，筛选上述基因都稳定插入基因组并能共同高表达的细胞，从而建立包装细胞系(packaging cell line)。之后，引入携带目的核酸片段的病毒基因组转录盒以构建携带目的核酸片段的病毒载体生产细胞系。如果使用逆转录后可以复制的非-SIN(self-inactivating)载体，这可以直接通过病毒感染来实现；否则，需要通过质粒化学转染并继之抗性筛选和克隆选择以获得在上述包装细胞基因组中稳定整合携带目的核酸片段的病毒基因组转录盒的生产细胞系。一种非常有效的方法是首先将含(可选择)标记基因的慢病毒基因组转录盒插入原始细胞中，并通过标记基因筛选稳定整合和高水平长效表达病毒载体的生产细胞系，之后再通过位点特异性重组酶(site-specific recombiase)，比如FLP-FRT或Cre-lox重组酶系统用目的核酸片段替换构建上述生产细胞系的标记基因来快速构建稳定生产携带目的核酸片段的病毒载体的生产细胞系。这种方法被证明允许建立高滴度的人源逆转录病毒载体生产细胞系(Schucht,R.,et al.(2006)."A new generation of retroviral producer cells:predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors." Mol Ther 14(2):285-292.；Loew,R.,et al.(2010)."A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration." Gene Ther 17(2):272-280.)。此外，利用类似原理可以开发目的核酸片段和/或外包膜蛋白快速替换的病毒载体包装细胞系或生产细胞系平台。稳定的逆转录病毒/慢病毒载体生产细胞系开发是一项耗时耗力的复杂系统工程，其需要一年或更长的时间来完全开发和表征细胞系平台，并且由于工作的复杂度，很多已经发表的工作因为产毒滴度、细胞系稳定性、培养适应等问题最终无法达到产业需求。但作为补偿，一旦研发成功获取稳定病毒生产细胞系，在临床和产业应用领域，稳定生产细胞系在工艺可靠性、工艺放大能力、生产成本和病毒产品安全性等方面，相较瞬时转染生产工艺具有不可替代的优势。首先，稳定生产细胞系生产工艺更加稳定，能提供完全表征的生产平台，以低的批次间差异生产更安全的病毒载体；其次，这种工艺更容易放大，不会像瞬时生产系统那样随着培养体积增大导致生产滴度快速下降；此外由于无需DNA质粒和转染试剂等原辅料，无需额外建立生产质粒的GMP生产线；最后，拥有更高的单位产量和更为简单的生产工艺质量控制。在扩大生产规模时，基于稳定生产细胞系的生产工艺在研发、生产、管理、运维和成本等方面会进一步凸显优势。这些优势对于促进基因治疗和细胞治疗领域的技术和药物产业化都是有益的。
利用稳定生产细胞系生产逆转录病毒载体在科研和临床实验领域已达十多年。然而稳定的慢病毒载体包装细胞系和生产细胞系的建立仍然是有挑战性的，原因在于多种HIV-1编码的蛋白质包括rev、tat、nef、env、vpr和PR蛋白酶具有细胞毒性能够导致细胞死亡，其中rev和PR蛋白酶在目前的包装系统 中仍然是必需的，而慢病毒系统常用的假型外包膜蛋白VSV-G也具有很强的细胞毒性。在实验室级别，通过质粒转染的瞬时生产是避免细胞毒性蛋白影响病毒制备的常用解决方法。但在开发慢病毒稳定生产细胞系时，上述有细胞毒性的蛋白的表达必须受到严格的控制比如使用诱导表达系统控制，否则所有高表达的细胞株都会死亡难以形成稳定生产细胞系。
目前能够在生产中获得的纯化前细胞原液中的逆转录病毒/慢病毒载体滴度为10 6至10 7感染颗粒/mL培养基。然而，在临床试验中治疗一名患者所需的载体的平均用量在10 10感染颗粒每患者这个级别。此外，病毒制备通常表征为低的感染颗粒/物理颗粒比(小于1:100)；同时这些病毒载体是非常敏感的，在37℃细胞培养上清中会快速地失去其感染性，半衰期约8-12小时，这进一步增加了对病毒产能的需求。据估计基于现有瞬转平台，每位患者需要约10-100L培养体积生产的病毒载体，而现有生产技术无论在滴度上还是在工艺放大上都难以再有本质的提高。因此，目前的逆转录病毒/慢病毒载体生产系统的表现是远低于治疗需求的，构建更高产的稳定生产细胞系并解决大规模生产工艺是解决产能问题的关键。
然而，构建逆转录病毒/慢病毒载体稳定生产细胞系具有相当大的技术难度。已有的稳定生产细胞系构建方法因为基因稳定插入效率低，通常需要引入大量的抗性基因进行同步或分步筛选，筛选过程很多在含血清贴壁培养条件下进行，筛选后细胞系不一定能适应无血清培养基悬浮培养条件，不符合国际上生物制药的技术发展趋势和安全性要求。并且在生产细胞系构建时，因为对各个逆转录/慢病毒包装基因的调控表达优化不足，使得最终稳定细胞系或者产毒滴度不高或者有高泄漏(leak)表达，而高泄露表达会导致生产细胞系不稳定且在细胞培养扩增时因包装基因泄露表达的细胞毒性影响细胞生长质量，难以达到工艺放大要求。
因此，本领域中目前迫切地需要一种高效的构建稳定的逆转录病毒和慢病毒载体生产细胞系的方法以及基于此方法构建的病毒包装/生产细胞系。
技术问题
本公开通过睡美人(Sleeping Beauty，SB)转座子系统结合PiggyBac(PB)转座子系统将病毒包装所需的gag、pol、rev(用于慢病毒载体)、env基因和携带目的核酸片段的病毒基因组转录盒稳定插入到宿主细胞(例如293T细胞)的基因组中并结合诱导表达系统(例如，Tet-On系统和/或Cumate系统)开发出可诱导调控的病毒生产系统，解决了上述问题。
技术解决方案
在使用逆转录病毒/慢病毒载体进行转导操作时，需要将目的核酸片段装载到包装进逆转录病毒/慢病毒的RNA基因组里。术语“目的核酸片段”一般根据应用目的可以是指基因，如编码蛋白的核酸序列；可以是有功能的核糖核酸(RNA)，如小分子干扰核糖核酸(small interfering RNA，简称siRNA)、长链非编码核糖核酸(long non-doding RNAs，简称LncRNA)，CRISPR基因编辑系统的导向RNA(guide RNA， gRNA)、转运核糖核酸(transfer RNA，简称tRNA)、核糖体核糖核酸(Ribosomal RNA，简称rRNA)或其他功能性核糖核酸的编码序列；可以是其它有功能核酸序列，如同源重组序列、能和蛋白结合的DNA或RNA序列、能和其它核酸片段(如引物或探针)结合的DNA或RNA序列；可以为任意一段来在自然界的核酸序列或人造核酸序列；也可以是以上一段或多段核酸序列的组合；目的核酸片段还可以包含调控基因表达的核酸序列如启动子、增强子、隔离子、聚腺苷酸信号等序列。逆转录病毒/或慢病毒的RNA基因组指在构建逆转录病毒/慢病毒载体时，能包装进病毒内的核糖核酸片段，一般包含病毒包装和转导的必要序列如ψ包装信号、长末端重复序列(long terminal repeat，LTR)。在慢病毒RNA基因组中，一般还包含全部5’端非翻译区域，300bp左右的5’端gag基因，中心多嘌呤束(cppt)片段，另外还带有rev应答元件(RRE)片段；缺少一段或多段以上片段可能会严重影响慢病毒的包装或转导效率。在制备逆转录病毒/慢病毒载体时，携带目的核酸片段的病毒基因组RNA片段一般通过构造携带目的核酸片段的病毒基因组转录盒(transcripiontal cassette)来实现，转录盒包含有启动子功能的核酸序列(可以是逆转录病毒/慢病毒自身LTR序列或其它异源启动子)，病毒RNA基因组对应的DNA序列，通常还有调控转录终止的聚腺苷酸信号序列。在制备逆转录病毒/慢病毒载体时，载有携带目的核酸片段的病毒基因组转录盒的构建体(比如将转录盒构建到转移载体质粒中)在被递送到宿主细胞后，通过宿主细胞的转录分子机制，转录成对应的能包装进病毒载体的携带目的核酸片段的病毒基因组RNA片段。
在本公开中，逆转录病毒包括，但不限于慢病毒，例如鼠白血病病毒(MLV)、人免疫缺陷病毒(HIV)、马传染性贫血病毒(EIAV)、小鼠乳房肿瘤病毒(MMTV)、Rous肉瘤病毒(RSV)、Fujinami肉瘤病毒(FuSV)、Moloney鼠白血病病毒(Mo-MLV)、FBR鼠骨肉瘤病毒(FBR MSV)、Moloney鼠肉瘤病毒(Mo-MSV)、Abelson鼠白血病病毒(A-MLV)、禽髓细胞瘤病病毒-29(MC29)、和禽脑脊髓炎病毒(AEV)、猿免疫缺陷病毒(Simian immunodeficiency virus,SIV)、猫免疫缺陷病毒(Feline immunodeficiency virus,FIV)、牛免疫缺陷病毒(Bovine immunodeficiency virus,BIV)、山羊关节炎-脑炎病毒(Caprine arthritis-encephalitis virus，CAEV)、长臂猿猿白血病病毒(Gibbon ape leukemia virus)、猫白血病病毒(Feline leukemia virus)、两性逆转录病毒(Amphotropic retrovirus)、嗜性逆转录病毒(Ecotropic retrovirus)、狒狒猿白血病病毒(Baboon ape leukemia virus)以及所有其他逆转录病毒科(Retroviridae)病毒。
转座子(transposon)是指可改变其在基因组内的位置的DNA序列。转座子可产生或逆转突变并改变细胞基因组的大小。DNA转座子在表达的转座酶(transpotase)作用下，可以以简单的剪切-粘贴的方式从一个DNA位点易位到另一个位点。转座是一个精确的过程，其中将限定的DNA片段，通常是转座子两端的直接重复序列(direct repeat，简称DR)和与它们相连的反向重复序列(invert repeat,简称IR)以及中间的插入序列(insert sequence,简称IS)，从一个DNA分子中切出并移动到相同或不同DNA分子或基因组中的另一个位点。
“睡美人(Sleeping Beauty，SB)转座子系统”由SB转座酶和转座子组成，其能够将特定的DNA插入序列插入脊椎动物的基因组中。SB转座酶可将转座子插入受体DNA序列中的TA二核苷酸碱基对中。插入位点可位于同一个DNA分子(或染色体)的其它位置或者位于另一个DNA分子(或染色体)中。在哺乳动物(包括人)基因组中，大约有2亿个TA位点。TA插入位点在转座子整合过程中复制。TA序列的这种复制是转座的标志并被用于在一些实验中确定机制。SB转座子由目的插入序列及位于其两端的用于被SB转座酶识别的IR/DR序列(本身含有短的直接重复(direct repeats，DR)的反向重复(inverted repeats，IR)，例如，如SEQ ID NO:23或其互补序列所示)组成。转座酶可在转座子内进行编码，或者转座酶可由另一个来源提供。SB转座子系统中的转座酶、IR/DR序列和转座子的野生型和不同变体在本领域中是已知的。已经公开的含睡美人转座子的质粒包括：pT/HB(Addgene，#26555)，pT2/HB(Addgene，#26557)，pT3(Yant,S.R.,et al.(2004)."Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase:critical residues for DNA binding and hyperactivity in mammalian cells."Mol Cell Biol 24(20):9239-9247.)等。SB转座酶的变体包括但不限于，SB10，SB11，SB100X(编码序列如SEQ ID NO:30所示)等(参见，例如，WO9840510A1、WO2003089618A2、WO2009003671A2、WO2017046259A1、WO2017158029A1等，上述文献通过引用结合与此)。在本文中，术语“睡美人(SB)转座子系统”可以包括含有SB转座酶的野生型和不同变体以及SB转座子的野生型和不同变体的SB转座子系统。
来源于粉纹夜蛾(Trichoplusia ni)的“PiggyBac(PB)转座子系统”由PB转座酶和转座子组成，其能够通过剪切-粘贴机制在载体和染色体之间进行有效转座。在转座过程中，PB转座酶识别位于转座子载体两端的转座子特异性反向末端反向重复序列(Inverted terminal repeart，简称ITR，3’ITR例如SEQ ID NO:24或其互补序列所示序列，5’ITR例如SEQ ID NO:25或其互补序列所示序列)，并且有效地移动来自原始位点的，在5’ITR和3’ITR之间的插入序列并有效地将其整合至染色体TTAA位点中。PiggyBac转座子系统的强大活性使得PB转座子载体中两个ITR之间的目的插入序列很容易地被移动到靶基因组中。PB转座子系统中的转座酶和转座子的野生型和不同变体(如ePiggyBac,编码序列如SEQ ID NO:31所示)在本领域中是已知的。关于PiggyBac转座子系统的信息可以参见，例如专利文献US6218185B1、US6551825B1、US7105343B1、US2002173634A1、US2010240133A1、WO2006122442、WO2010085699、WO2010099296、WO2010099301、WO2012074758、US8592211等，以及非专利文献Lacoste,A.,et al.(2009)."An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells."Cell Stem Cell 5(3):332-342；Yusa,K.,et al.(2011)."A hyperactive piggyBac transposase for mammalian applications."Proc Natl Acad Sci U S A 108(4):1531-1536(上述文献通过引用结合与此)。在文中，术语“PiggyBac(PB)转座子系统”可以包括含有PB转座酶的野生型和不同变体以及PB转座子的野生型和不同变体的PB转座子系统。
在本公开中，通过诱导表达系统(Inducible expression system)来控制转入宿主细胞中的核酸构建体中的相关基因的转录和表达。在本公开中，可用的诱导表达系统的实例包括，但不限于，四环素诱导系统中的Tet-Off诱导表达系统(参见，例如，Gossen,M.and H.Bujard(1992)."Tight control of gene expression in mammalian cells by tetracycline-responsive promoters."Proc Natl Acad Sci U S A 89(12):5547-5551；Yu,H.,et al.(1996)."Inducible human immunodeficiency virus type 1packaging cell lines."J Virol 70(7):4530-4537；Kaul,M.,et al.(1998)."Regulated lentiviral packaging cell line devoid of most viral cis-acting sequences."Virology 249(1):167-174(上述文献通过引用结合与此))、四环素诱导系统中的Tet-On诱导表达系统(参见，例如，WO0075347A2、WO2007058527A2(上述文献通过引用结合与此))、Cumate系统(参见，例如，WO02088346A2、WO2006037215A1(上述文献通过引用结合与此))、Cumate诱导表达系统(参照：Mullick,A.,et al.(2006)."The cumate gene-switch:a system for regulated expression in mammalian cells."BMC Biotechnol 6:43；WO02088346A2、WO2006037215A1(上述文献通过引用结合与此))或其组合。在一个方面，用于可诱导表达的诱导表达系统是Tet-On诱导表达系统或Cumate诱导表达系统。在另一个方面，用于可诱导表达的诱导表达系统是Tet-On诱导表达系统与Cumate诱导表达系统的组合。在Tet-On诱导表达系统中，四环素依赖的反式激活物(reverse tetracycline controlled transactivator，rtTA)仅在存在四环素或多西环素(Dox)等四环素衍生物的情况下才能够结合Tet-On诱导表达系统TRE响应元件(含有多拷贝连续TetO操作子序列和最小启动子序列的核酸序列，Tet Response Element，下文称为TRE)以启动下游连接的被调控核酸片段的转录。目前常用的Tet-On诱导表达系统包括第二代Tet-On诱导表达系统(Tet-On Advanced，Clontech，其中的反式激活物rtTA和响应元件TRE在下文中分别称为rtTA adv(编码核酸序列：SEQ ID NO:29)和TRE adv(SEQ ID NO:21)和第三代Tet-On诱导表达系统(Tet-On3G，Clontech，其中的反式激活物rtTA和响应元件TRE在下文中分别称为rtTA 3G(编码核酸序列：SEQ ID NO:28)和TRE 3G(SEQ ID NO:19)。第二代和第三代Tet-On诱导表达系统中的反式激活物rtTA和TRE可以组合使用，例如rtTA adv可以组合TRE 3G使用，rtTA 3G也可以组合TRE adv使用。Cumate诱导表达系统(Cumate Inducible System)开发自Pseudomonas putida的p-cym操纵子，由在启动子TATA盒下游连接的CuO操作子和Cumate及其衍生物依赖的能和CuO操作子结合的阻遏物CymR蛋白组成。在无Cumate的条件下，CymR蛋白和CuO操作子结合，抑制下游连接的目的核酸序列的转录表达；当CymR蛋白和Cumate结合后，其和CuO操作子的亲和力下降并分离不再抑制下游连接的目的核酸序列的转录表达。在本公开中，在将Tet-On诱导表达系统与Cumate诱导表达系统组合使用的情况下，构建了包含多拷贝连续TetO操作子序列、含有TATA盒的最小启动子序列以及CuO操作子序列的复合响应元件序列。其中，基于第二代Tet-On诱导表达系统中TRE adv序列中多拷贝连续TetO操作子序列设计了TRE advCuO(SEQ ID NO:22)复合响应元件；基于第三代Tet-On诱导表达系统中TRE 3G序列中多拷贝连续TetO操作子序列设计了TRE 3GCuO复合响应元件(SEQ ID NO:20)。在将Tet-On诱导表达系统与 Cumate诱导表达系统组合使用的情况下，需要同时表达Tet-On诱导系统的反式激活物rtTA蛋白和Cumate诱导系统的阻遏物CymR蛋白，其中使用的Tet-On反式激活物可以是rtTA adv，也可以是rtTA 3G。优选地，使用基于人编码密码子优化的rtTA 3G(SEQ ID NO:28)和CymR(SEQ ID NO:27)序列。在一个方面，可用于本公开目的的诱导表达系统还可以包括金属离子诱导型、激素诱导型和温度诱导型诱导表达系统。
在本公开的一个方面中，gag和pol基因(实施例中以gag/pol表示gag基因和pol基因位于同一构建体，且通过移码(frameshift)方式翻译表达)、rev基因、病毒包膜蛋白(例如VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒中的一种或多种的转录是受控的，所述受控的转录是通过将所述基因或序列置于诱导表达系统的控制下实现的，优选地，所述受控的转录是通过将所述基因或序列置于Tet-On和/或Cumate诱导表达系统的控制下实现的，更优选是在Tet-On和Cumate诱导表达的双重控制下或是在第三代Tet-On诱导表达系统的单独调控下。在本公开的一个方面中，gag和pol基因、rev基因、病毒包膜蛋白的编码序列和携带目的核酸片段的病毒基因组转录盒的转录全部都是受控的，所述受控的转录是通过将所述基因或序列置于诱导表达系统的控制下实现的，优选地，所述受控的转录是通过将所述基因或序列置于Tet-On和/或Cumate诱导表达系统的控制下实现的，更优选是在Tet-On和Cumate诱导表达系统的双重控制下或是在第三代Tet-On诱导表达系统的单独调控下。在本公开的一个方面中，gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒中的rev基因和病毒包膜蛋白(VSV-G)的编码序列的转录是受控的，所述受控的转录是通过将所述基因或序列置于诱导表达系统的控制下实现的，优选地，所述受控的转录是通过将所述基因或序列置于Tet-On和/或Cumate诱导表达系统的控制下实现的，更优选是在Tet-On和Cumate诱导表达的双重控制下或是在第三代Tet-On诱导表达系统的单独调控下。
被调控核酸片段在诱导条件下的表达量和非诱导条件下的泄露表达量是评价诱导表达系统优劣的两个重要指标，好的诱导表达系统需要具有更高的诱导表达量和更低的泄露表达量。本发明人发现，单独的Tet-On诱导表达系统或Tet-On和Cumate复合诱导表达系统的诱导表达量和泄露表达量受(1)TRE响应元件中最小启动子序列；(2)rtTA反式激活物的突变体的选择；(3)诱导系统响应元件3’端和被调控的目的核酸序列5’端之间是否连接可剪切的内含子序列影响。本公开共设计和使用TRE adv、TRE 3G、TRE advCuO和TRE 3GCuO四种响应元件，以及rtTA adv和rtTA 3G两种反式激活物。第三代TRE 3G响应元件比第二代TRE adv响应元件控制更严格，被调控核酸片段有更低的泄露表达量，但诱导表达量低于TRE adv。本公开设计的Tet-On和Cumate复合诱导表达系统响应元件TRE advCuO(SEQ ID NO:22)和TRE 3GCuO(SEQ ID NO:20)比任意单Tet-On诱导系统响应元件控制更严格，显著降低了被调控核酸片段的泄露表达量，但同时也会显著降低被调控核酸片段的诱导表达量。反式激活物rtTA 3G和rtTA adv相比，在诱导条件下，有相似的转录激活活性；在无诱导条件下，rtTA 3G的基础泄露转录激活活性更低。 在表达质粒中启动子3’端连接的内含子一般可以提高转录信使RNA(mRNA)的稳定性并提高mRNA的转运出细胞核的效率(参照Akef,A.,et al.(2015)."Splicing promotes the nuclear export of beta-globin mRNA by overcoming nuclear retention elements."RNA 21(11):1908-1920)并提高被调控核酸片段的表达量。本发明人发现，在单一诱导表达系统中的响应元件3’端和被调控核酸片段5’端之间连接可剪切的内含子能提高被调控核酸片段的诱导表达量，但同时也会显著增加被调控核酸片段的泄露表达量。在本公开的Tet-On和Cumate复合诱导系统中，在响应元件3’端和被调控核酸片段5’端之间插入可剪切的内含子序列可以在不显著增加无诱导条件下泄露表达量的同时显著提高在诱导条件下的诱导表达量。
在构建慢病毒生产细胞系时，需要平衡rev基因、VSV-G基因、gag和pol基因以及携带目的核酸片段的病毒基因组转录盒的表达量、表达量的相对比例、表达的蛋白或转录的核酸的细胞毒性以及表达或转录的先后顺序，因此需要对每一个基因和携带目的核酸片段的病毒基因组转录盒的启动子或诱导表达系统响应元件进行优化。在需要表达的慢病毒rev基因、VSV-G基因、gag和pol基因以及携带目的核酸片段的病毒基因组转录盒中，rev和膜蛋白VSV-G有明显细胞毒性，并且rev还调控通常含有RRE序列的gag和pol基因的表达，所以需要用诱导表达系统严格控制。Gag和pol基因的表达在rev受控的条件下并非必须严格调控，但用诱导表达系统控制gag和pol基因的表达可能进一步降低无诱导条件下泄露表达的病毒滴度，尤其是在单Tet-On诱导表达系统控制rev和VSV-G的表达情况下。基于此，本公开基于4种响应元件以及响应元件3’端和被调控核酸片段5’端之间是否连接有可剪接的内含子序列设计并测试了8种慢病毒基因表达的调控序列：TRE adv(诱导系统响应元件为TRE adv序列(SEQ ID NO:21)，并且响应元件3’端和被调控核酸片段5’端之间不存在内含子)、TRE advCuO(诱导系统响应元件为TRE advCuO序列(SEQ ID NO:22)，并且响应元件3’端和被调控核酸片段5’端之间不存在内含子)、TRE 3G(诱导系统响应元件为TRE 3G序列(SEQ ID NO:19)，并且响应元件3’端和被调控核酸片段5’端之间不存在内含子)、TRE 3GCuO(诱导系统响应元件为TRE 3GCuO序列(SEQ ID NO:20)，并且响应元件3’端和被调控核酸片段5’端之间不存在内含子)、TRE adv-内含子(诱导系统响应元件为TRE adv序列，并且响应元件3’端和被调控核酸片段5’端之间连接可剪切的内含子)、TRE advCuO-内含子(诱导系统响应元件为TRE advCuO序列，并且响应元件3’端和被调控核酸片段5’端之间连接可剪切的内含子)、TRE 3G-内含子(诱导系统响应元件为TRE 3G序列，并且响应元件3’端和被调控核酸片段5’端之间连接可剪切的内含子)和TRE 3GCuO-内含子(诱导系统响应元件为TRE 3GCuO序列，并且响应元件3’端和被调控核酸片段5’端之间连接可剪切的内含子)，并以此优化了调控慢病毒各个基因表达的最优组合。在本公开中，在响应元件3’端和受响应元件控制的被调控核酸片段5’端之间的内含子可以与所述响应元件和所述受响应元件控制的被调控核酸片段直接相连，也可以不直接相连，这是可以由本领域技术人员根据本公开的内容并且根据需要调整的，并且在不直接相连的情况下，本领域技术人员可以理解，响应元件和受响应元件控制的被调控核酸片段之间的距离不应影响响应元件对被调控核酸片段的转录的控制。在本公开的一个方 面，在单Tet-On诱导调控系统的情况下，rev的调控序列优选为TRE 3G；VSV-G的调控序列优选为TRE 3G-内含子；gag和pol的调控序列优选为CMV启动子(或其他本领域中常用的真核启动子)-内含子或TRE 3G-内含子，并且更优选为TRE 3G-内含子；rtTA反式激活物优选rtTA 3G。在此条件下，瞬时转染上述相关质粒可以达到6.94E+05 RLU和176倍诱导/泄露产毒滴度比值；相较第二代Tet-On系统(4.64E+05 RLU，7倍诱导/泄露产毒滴度比值)在产毒滴度上提高了49.6％，在控制病毒泄露表达上提高了25倍。在本公开的一个方面，在Tet-On和Cumate复合诱导调控系统的情况下，rev的调控序列优选为TRE advCuO、TRE advCuO-内含子、TRE 3G、TRE 3GCuO、和TRE 3GCuO-内含子，更优选TRE 3G、TRE 3GCuO和TRE 3GCuO-内含子，再进一步优选TRE 3G；VSV-G的调控序列优选TRE advCuO、TRE advCuO-内含子、TRE 3G-内含子和TRE 3GCuO-内含子，更优选TRE advCuO-内含子、TRE 3G-内含子和TRE 3GCuO-内含子，再进一步优选TRE 3GCuO-内含子；gag和pol的调控序列优选带有内含子的调控序列，包括CMV启动子(或其他本领域中常用的真核启动子)-内含子、TRE adv-内含子、TRE advCuO-内含子、TRE 3G-内含子和TRE 3GCuO-内含子，更优选TRE 3GCuO-内含子；rtTA反式激活物优选rtTA 3G。在此条件下，瞬时转染上述相关质粒可以达到1.46E+06 RLU和12032倍诱导/泄露产毒滴度比值；相较第二代Tet-On系统(4.64E+05 RLU，7倍诱导/泄露产毒滴度比值)在产毒滴度上提高了214.7％，在控制病毒泄露表达上提高了1719倍。
上述在响应元件3’端和被调控核酸片段5’端之间连接的可剪接的内含子序列的选择并无特定限制，本领域技术人员可以理解，凡是在哺乳动物细胞中能进行RNA剪接(Splicing)的序列都能实现上述功能。可以选择的内含子序列包括但不限于常用的克隆载体上的内含子如：兔β-球蛋白内含子、源自人β-球蛋白和免疫球蛋白重链内含子的杂合内含子、EF-1α内含子A、SV40内含子、源自腺病毒和免疫球蛋白重链内含子的杂合内含子、修饰的人巨细胞病毒内含子、源自鸡β-肌动蛋白(CBA)和小鼠微小病毒(MMV)内含子的杂合内含子、源自鸡β-肌动蛋白和兔β-球蛋白内含子的嵌合物和mP1内含子；也可以是任意真核生物任意基因的任意内含子；或者是基于内含子剪接规则设计的人造内含子序列。本公开实施例使用的内含子序列可以例如是如SEQ ID NO:26所示的人β-球蛋白内含子序列(BGI序列)。
在本公开中，利用转座子系统将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒稳定整合进宿主细胞基因组中。在本公开的一个方面，当将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和/或携带目的核酸片段的病毒基因组转录盒的一种或多种置于Tet-On和/或Cumate诱导表达系统的控制下时，利用转座子系统将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒以及Tet-On反式激活物rtTA蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列整合进宿主细胞基因组中。在本公开的一个方面，利用SB转座子系统或PB转座子系统将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列、携带目的核酸片段的病毒基因组转录盒以及诱导表达系统激活物或阻遏物蛋白编码序列(例如，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物蛋白CymR的编码序 列)一次性整合进宿主细胞基因组中，直接构建制备慢病毒的生产细胞系。在本公开的一个方面，利用SB转座子系统和PB转座子系统分别将上述基因和序列转入宿主细胞中。在本公开的一个优选方面，将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒(以及，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)分两次整合进宿主细胞基因组中。第一次使用SB转座子系统或PB转座子系统中的任一种转座子系统将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒(以及，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)中的一种或多种整合进宿主细胞基因组中构建包装细胞系，第二次使用SB转座子系统或PB转座子系统中的另一种未使用的转座子系统将gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒(以及，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)中剩余的一种或多种整合进上述包装细胞系基因组中进而构建生产细胞系。在本公开一个优选的方面，首先使用SB转座子系统或PB转座子系统中的任一种转座子系统将gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)整合进宿主细胞基因组中构建包装细胞系，然后使用SB转座子系统或PB转座子系统中的另一种未使用的转座子系统再将携带目的核酸片段的病毒基因组转录盒整合进已经在基因组中整合了gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)的包装细胞系基因组中进而构建生产细胞系。在此种情况下，可以仅通过向已经构建好的基因组整合有gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列)的稳定的包装细胞转入单一的携带目的核酸片段的病毒基因组转录盒的构建体并进行简单的筛选在基因组中稳定整合上述构建体的细胞克隆就可以构建得到可以用于制备携带目的核酸片段的慢病毒载体的慢病毒载体生产细胞系。本发明人发现，如果仅使用同一种转座子系统(仅使用SB系统或仅使用PB系统)将全部上述基因和序列依次整合进宿主细胞基因组中，则在转入后一种或多种基因或序列时，转座酶可能对已转入宿主细胞中并整合在宿主基因组中的前一种或多种基因或序列起作用，从而导致所述前一种或多种基因或序列被从宿主基因组上剪切下来，从而降低制备包含全部基因和序列的包装/生产细胞系的生产效率。本发明的发明人发现在两次将一种或多种基因或序列整合进宿主细胞基因组时使用不同的转座子系统可以有效地避免上述不利作用的发生。因此，在本公开中，所述两次将一种或多种基因或序列整合进宿主细胞基因组过 程中使用不同的转座子系统，即例如，首先使用SB系统将上述一种或多种基因或序列整合进宿主细胞基因组中，然后使用PB系统将剩余的一种或多种基因或序列整合进宿主细胞基因组中，或首先使用PB系统将上述一种或多种基因或序列整合进宿主细胞基因组中，然后使用SB系统将剩余的一种或多种基因或序列整合进宿主细胞基因组中。在本公开一个优选的方面，首先使用SB系统将gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)整合进宿主细胞基因组中构建包装细胞，然后再使用PB系统将携带目的核酸片段的病毒基因组转录盒整合进已经在基因组中稳定整合有gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)的包装细胞的基因组中进而构建生产细胞。
区别于传统的转染筛选法构建稳定转染细胞系，PB和SB转座子系统均具有主动将插入序列片段整合到宿主细胞基因组中的功能。通过优化，单次瞬时转染在无抗性筛选条件下细胞群中平均成功稳定整合效率可达40％以上，可以在一次转染操作中获得在细胞基因组中稳定整合20个拷贝以上插入序列片段的细胞克隆，并且在一次操作中便可将多种不同大小、序列不同的插入序列片段多拷贝的整合进细胞的基因组中。在另一方面，使用PB和SB转座子系统将插入序列片段整合进宿主细胞基因组的效率和在细胞内引入各转座子构建体时的总转座子构建体和转座酶构建体的摩尔比例，转座子构建体的引入量和转座子中插入序列片段的长短有关。一般情况下转座子的构建体可以是DNA质粒、微环DNA(minicircle DNA)、线性DNA片段或病毒载体；转座酶的构建体可以是DNA质粒、微环DNA(minicircle DNA)、线性DNA片段、病毒载体、RNA或蛋白。在转座子和转座酶使用DNA质粒做为构建体时，提高转座子/转座酶质粒在瞬时转染时的摩尔比例至5:1或以上可以显著提高插入序列片段在细胞基因组中的稳定整合效率。另外转座子在细胞基因组中的整合效率随着插入序列片段增长而降低，两者存在较为稳定的线性关系，在细胞基因组中同时整合多种插入序列片段的操作中可以根据转座子插入序列片段的长短调整各转座子构建体的加入量和相对摩尔比例来控制最后各插入序列片段整合进细胞基因组中的拷贝数和摩尔比例。在本公开的一个方面，优选地首先使用SB转座子系统将gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)整合进宿主细胞基因组中。在构建过程中，通过瞬时转染含有上述基因和蛋白编码序列的转座子质粒和SB转座酶质粒，在总转座子质粒和SB转座酶质粒摩尔比5:1以上，更优选的在10:1至40:1的条件下构建上述包装细胞系。所构建的包装细胞系基因组中稳定整合至少一个拷贝的gag和pol基因、rev基因和病毒包膜蛋白(VSV-G)的编码序列(优选地，以及至少一个拷 贝的诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，至少一个拷贝的Tet-On反式激活物蛋白的编码序列和/或至少一个拷贝的Cumate操纵子的阻遏物CymR蛋白的编码序列)，优选地调整gag和pol基因整合进宿主细胞基因组的拷贝数在2个拷贝数每细胞以上；gag和pol基因序列和VSV-G蛋白编码序列整合进宿主细胞基因组的拷贝比例在1:1至3:1之间；更优选地调整gag和pol基因整合进宿主细胞基因组的拷贝数在4至6个拷贝数每细胞；gag和pol基因序列和VSV-G蛋白编码序列整合进宿主细胞基因组的拷贝比例在2:1至3:1之间。在通过SB转座子系统构建上述包装细胞系后，再使用PB转座子系统将携带目的核酸片段的病毒基因组转录盒整合进上述包装细胞系的基因组中。在构建过程中，通过瞬时转染含有携带目的核酸片段的病毒基因组转录盒的转座子质粒和PB转座酶质粒，在转座子质粒和PB转座酶质粒摩尔比5:1以上，更优选的在10:1至40:1的条件下构建稳定慢病毒生产细胞系。所构建的生产细胞系基因组中稳定整合至少一个拷贝的携带目的核酸片段的病毒基因组转录盒。这种使用PB和SB双转座子系统构建生产细胞系的方式具有提高插入基因拷贝数，准确调整整合进宿主细胞基因组中各插入序列片段的摩尔比例，提高构建筛选最优高产生产细胞系成功率，减少细胞筛选步骤和筛选时间，减少抗性基因的使用数目等明显优势。
在本公开的一个方面中，gag基因、pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列、携带目的核酸片段的病毒基因组转录盒以及诱导表达系统激活物和/或阻遏物蛋白编码序列(例如，Tet-On反式激活物rtTA蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)分别被构建在不同的转座子构建体上；在本公开的一个方面中，以上基因或序列片段的任意两种被构建在一个转座子构建体上；在本公开的一个方面中，以上基因或序列片段的任意三种被构建在一个转座子构建体上；在本公开的一个方面中，以上基因或序列片段的任意四种被构建在一个转座子构建体上；在本公开的一个方面中，以上基因或序列片段的任意5种被构建在一个转座子构建体上；在本公开的一个方面中，以上基因或序列片段的任意六种被构建在一个转座子构建体上；在本公开的一个方面中，以上基因或序列片段全部被构建在一个转座子构建体上。为降低生产细胞系产生具有复制能力的慢病毒(replication-competent lentivirus,简称RCL)的可能性，并且为方便优化各病毒包装基因和序列片段整合在宿主细胞基因组中的拷贝数比例，优选地，将gag基因和pol基因构建在同一个转座子构建体上；将rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒分别构建在三个独立转座子构建体上；在单独使用Tet-On诱导表达系统的情况下，将Tet-On反式激活物rtTA蛋白的编码序列构建在一个独立转座子构建体上；在使用Tet-On和Cumate复合诱导表达系统的情况下，Tet-On反式激活物rtTA蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列优选构建在同一个转座子构建体上。
在本公开中，分别携带gag和pol基因、rev基因、病毒包膜蛋白(VSV-G)的编码序列和携带目的核酸片段的病毒基因组转录盒以及诱导表达系统激活物和/或阻遏物蛋白编码序列(例如，Tet-On反式激活物rtTA蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)的构建体中的一种或多种 还携带有筛选基因序列。在本公开中，所述筛选基因序列是用于真核细胞的筛选基因序列。在本公开的一个方面，所述筛选基因可以例如选自选自：潮霉素抗性基因、嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、博莱霉素抗性基因；或所述筛选基因是代谢通路筛选基因，例如选自编码二氢叶酸还原酶(Dihydrofolate reductase)、谷氨酰胺合成酶(Glutamine synthetase)、胸苷激酶(Thymidine kinase)的核酸序列。在本公开的一个方面，所述筛选基因的表达可以通过启动子、内部核糖体进入位点(internal ribosome entry site,简称IRES)或P2A自剪切多肽序列调控表达，优选地通过SV40启动子调控表达。在本公开的一个方面，在首先使用SB转座子系统将gag和pol基因、rev基因和病毒包膜蛋白(例如VSV-G)的编码序列(优选地，以及诱导表达系统激活物和/或阻遏物蛋白编码序列，例如，在使用Tet-On和/或Cumate诱导表达系统的情况下，Tet-On反式激活物rtTA蛋白的编码序列和/或Cumate操纵子的阻遏物CymR蛋白的编码序列)整合进宿主细胞基因组构建包装细胞系的操作步骤中，所述构建体中的一种或多种可以携带不同的筛选基因序列，优选地，仅使用一种筛选基因序列，其可以位于以上任一种构建体上，例如其可以位于携带诱导表达系统激活物和/或阻遏物蛋白编码序列的构建体上(例如，在单独使用Tet-On诱导表达系统的情况下，Tet-On反式激活物rtTA蛋白的编码序列的构建体中携带一种筛选基因序列(例如，潮霉素抗性基因(HygroR))；或在使用Tet-On和Cumate复合诱导表达系统的情况下，Tet-On反式激活物rtTA蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列所在的同一构建体中携带一种筛选基因序列(例如，潮霉素抗性基因(HygroR))。之后在使用PB转座子系统将携带目的核酸片段的病毒基因组转录盒稳定整合进上述包装细胞系基因组中构建生产细胞系的操作步骤中，所述携带目的核酸片段的病毒基因组转录盒的构建体优选地携带与之前不同的另一种抗性基因序列(例如，嘌呤霉素抗性基因(PuroR))。在本公开的一个方面，病毒包膜蛋白(VSV-G)的编码序列的构建体上携带与上述两种不同的第三种抗性基因序列(例如，杀稻瘟菌素抗性基因(BSD))。在本公开的一个方面，在足够高通量单克隆细胞筛选的条件下，本公开所述各构建体中可以不含有任何筛选基因，仅通过单克隆细胞产毒能力筛选稳定高产包装/生产细胞系。
在本公开中，携带目的核酸片段的慢病毒基因组转录盒可以来自第二代慢病毒载体的转移载体质粒例如pLVPRT-tTR-KRAB(Addgene，#11648)、pLenti CMVtight eGFP Puro(w771-1)(Addgene，#26431)或第三代慢病毒载体的转移载体质粒例如pSLIK-Hygro(Addgene，#25737)、pHIV-EGFP(Addgene，#21373)、pSico(Addgene，#11578)、pRRLSIN.cPPT.PGK-GFP.WPRE(Addgene，#12252)、Tet-pLKO-puro(Addgene，#21915)、pLenti-puro(Addgene，#39481)、pLVUT-tTR-KRAB(Addgene，#11651)等。第三代慢病毒载体和第二代慢病毒载体的病毒基因组转录盒大多都共有LTR、5’端非编码片段、HIV-1Ψ包装信号、RRE、cPPT和gag部分序列等在病毒包装和转导过程中起关键作用的核酸序列，第三代相比第二代慢病毒基因组转录盒主要区别是用组成型活性启动子如CMV或RSV替代5’LTR序列中起启动子功能的U3序列，并删除了3’-LTR序列的中U3序列，使慢病毒转移载体成为了SIN(self-inactivating) 载体。在第三代慢病毒基因组转录盒中，pSLIK-Hygro、pHIV-EGFP、pSico载体‘使用CMV启动子转录慢病毒基因组RNA，而pRRLSIN.cPPT.PGK-GFP.WPRE、Tet-pLKO-puro、pLenti-puro使用RSV启动子转录慢病毒基因组RNA。Tet-pLKO-puro和pLenti-puro与其它第三代慢病毒转移载体质粒相比，在慢病毒基因组转录盒中不含有WPRE序列。上述慢病毒转移载体质粒中的慢病毒基因组转录盒在原理上都适用本公开所描述的构建稳定慢病毒生产的细胞系方法。在本公开的一个方面，基于pRRLSIN.cPPT.PGK-GFP.WPRE(Addgene，#12252)转移载体质粒中的核酸序列设计本公开所用慢病毒基因组转录盒的序列以及构建了含有此序列的质粒构建体。
本公开所述的逆转录病毒/慢病毒生产细胞系构建方法同样可以构建用于快速替换目的核酸序列和/或外包膜蛋白的病毒生产细胞系，包括但不限于使用位点特异性重组(site-specific recombination)策略构建的快速替换病毒生产细胞系。位点特异性重组也叫做保守的位点特异性重组(conservative site-specific recombination)，是一种DNA链交换发生在至少一定程度的同源序列片段之间的基因重组。许多不同的基因组修饰策略，比如重组酶介导的表达盒交换(recombinase-mediated cassette exchange,简称RMCE)，都依赖于位点特意性重组，这是一种将转录盒或目的核酸片段靶向导入预定基因组特定位点的先进方法。位点特异性重组一般由位点特异性重组酶(site-specific recombiase,简称SSR)和被其识别的，并发生同源性重组的特定位点序列组成。基于氨基酸序列同源性和重组酶催化机制的相关性，多数位点特异性重组酶可以分类为酪氨酸(Tyr)重组酶或丝氨酸(Ser)重组酶。常见的酪氨酸重组酶有Cre或FLP；常见的丝氨酸重组酶有gamma-delta和Tn3分解酶等经典成员以及新发现的φC31-,Bxb1-,and R4整合酶(integrase)。结合位点特异性重组酶并发生同源性重组的特定位点序列长度一般为30至200个碱基，由和位点特异性重组酶结合的两段部分反向重复对称序列以及被其包围的发生同源重组的中间序列组成。一般位点特异性重组发生在两段相同的特定位点序列，但也有例外(如在λ噬菌体整合酶系统的attP和attB位点)。Cre-lox和FLP-FRT位点特异性重组系统是用于重组酶介导的表达盒交换的常用成熟系统。Cre-lox系统由Cre重组酶和Cre识别的loxp序列组成；FLP-FRT系统由flippase重组酶(FLP)和FLP识别的短反转酶识别位点序列(short flippase recognition target，简称FRT)组成。这两种位点特异性重组系统基于其位点特异性重组酶识别位点序列的数目、方向、在同一或不同DNA分子以及识别位点的核酸突变设计策略，可以实现目标核酸序列片段的切除，插入、易位和反转。在设计重组酶介导的表达盒交换策略时，通常在要被替换的核酸序列(本身可以含有标记基因或连接标记基因(第一标记基因))的两端设有位点特异性重组酶识别序列(第一标记基因与被替换目的核酸序列都位于两段特异性重组酶识别位点序列之间)，并基于第一标记基因的特性筛选细胞系。待替换目的核酸序列(优选地连接有不同于第一标记基因的第二标记基因)一般被设置在载体上(比如质粒或病毒载体)，其两端同样具有位点特异性重组酶识别序列(第二标记基因与待替换目的核酸序列都位于两段特异性重组酶识别位点序列之间)。当携带待替换目的核酸片段的载体和相对应的位点特异性重组酶表达载体(如携带编码位点特 异性重组酶的质粒、病毒载体、RNA或位点重组酶蛋白)同时被递送到基于第一标记基因筛选的细胞系时，在位点特异性重组酶的催化下，原有的要被替换的核酸片段被带有第二标记基因的待替换目的核酸片段替换。在经过基于第二标记基因特性的筛选后，可以获取成功完成重组酶介导的表达盒交换的目的细胞系。使用类似策略可以构建用于快速替换目的核酸片段和/或外包膜蛋白的快速替换病毒生产细胞系。例如，先基于原有核酸片段(本身可以含有标记基因或连接标记基因(第一标记基因A))和/或连接有标记基因(第一标记基因B)的原有外包膜蛋白的编码序列根据本公开的方法构建快速替换病毒生产细胞系，所述原有核酸片段(本身可以含有标记基因或连接有标记基因)的两端设有位点特异性重组酶识别位点序列，和/或连接有标记基因的原有外包膜蛋白的编码序列的两端设有位点特异性重组酶识别位点序列。在快速替换目的核酸片段和/或外包膜蛋白的编码序列时，将包含两端具有特异性重组酶识别位点序列并且与标记基因(第二标记基因)相连的待替换的目的核酸片段和/或待替换的外包膜蛋白的编码序列(标记基因(第二标记基因A)与待替换的目的核酸片段都位于两段特异性重组酶识别位点序列之间，和/或标记基因(第二标记基因B)和待替换的外包膜蛋白的编码序列都位于两段特异性重组酶识别位点序列之间，与待替换序列连接的第二标记基因(A和/或B)和已经构建生产细胞系使用的第一标记基因(A和/或B)不同)的载体和相对应的位点特异性重组酶表达载体同时递送到上述快速替换病毒生产细胞系中，并基于与待替换的目的核酸片段和/或待替换的外包膜蛋白的编码序列连接的第二标记基因(A和/或B)筛选成功完成重组酶介导的表达盒交换的能生产含有新的目的核酸片段和/或新的外包膜蛋白的病毒生产细胞系。以上所述的标记基因例如是用于真核细胞的抗性基因序列，所述抗性基因序列例如选自：潮霉素抗性基因、嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、博莱霉素抗性基因；或所述标记基因例如是代谢通路筛选基因，所述代谢通路筛选基因例如选自编码二氢叶酸还原酶(Dihydrofolate reductase)、谷氨酰胺合成酶(Glutamine synthetase)、胸苷激酶(Thymidine kinase)的基因；或所述标记基因例如是荧光蛋白标记基因，所述荧光蛋白标记基因例如选自编码绿色荧光蛋白(EGFP)、红色荧光蛋白(dsRed)、樱桃色荧光蛋白(mcherry)、青色荧光蛋白(ECFP)、黄色荧光蛋白(EYFP)、以及荧光蛋白的其它突变衍生蛋白的基因序列；或所述标记基因例如是能够用于报告基因检测蛋白酶基因，所述能够用于报告基因检测蛋白酶基因例如选自编码萤光素酶(luciferase)、β-半乳糖苷酶(β-galactosidase)、氯霉素乙酰转移酶(chloramphenical acetyltransferase)的基因序列；或所述标记基因例如是以上各种标记基因的任意组合。
在本公开中，可用于构建逆转录病毒/慢病毒包装细胞系和生产细胞系的宿主细胞是哺乳动物细胞。适合在本公开中使用的宿主细胞的实例有293T细胞、HepG2细胞、CHO细胞、BHK细胞、HEK293细胞、COS细胞、NIH/3T3细胞、Vero细胞、HT1080细胞、Te671细胞、CEM细胞、NSO细胞或PerC6细胞，以及以上细胞来源的衍生细胞。在一个方面，所述宿主细胞是HEK293细胞或来源于HEK293细 胞的细胞。在一个方面，所述宿主细胞是293T细胞。在一个方面，生产/包装细胞可以被贴壁培养或悬浮培养。在一个方面，生产/包装细胞可以在有血清或无血清添加的条件下培养。
在本公开中，可用于Tet-On诱导表达系统的四环素及其衍生物包括在结构上与四环素相似的化合物，其能够与本公开所述四环素依赖的反式激活物rtTA相结合，其结合常数Ka至少达到10 -6M；优选地，其结合常数Ka达到或强于10 -9M。四环素衍生物例如可以选自：多西环素(Dox)、脱水四环素(Atc)、氯四环素、土霉素和脱氧四环素。
在本公开中，可用于Cumate诱导表达系统的，与阻遏物CymR相结合的Cumate功能类似物例如可以选自：对乙基苯甲酸、对丙基苯甲酸、对异丙基苯甲酸、对异丁基苯甲酸、对叔丁基苯甲酸,对正二甲基氨基苯甲酸,对正乙基氨基苯甲酸。以及例如在美国专利号7,745,592中描述的其他Cumate功能类似物。
在本公开的一个方面，提供以下各项：
第1项：一种用于制备用于生产携带目的核酸片段的逆转录病毒载体的生产细胞的方法，所述方法包括：
利用睡美人(Sleeping Beauty，SB)转座子系统将逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用PiggyBac(PB)转座子系统将所述逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用SB转座子系统将所述逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中。
第2项：一种用于制备用于生产携带目的核酸片段的慢病毒载体的生产细胞的方法，所述方法包括：
利用SB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用PB转座子系统将所述慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用SB转座子系统将所述慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中。
第3项：根据第2项所述的方法，其中利用SB转座子系统将慢病毒的gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用PB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将慢病毒的gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用SB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中。
第4项：根据第2项所述的方法，其中所述慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列位于两个以上的构建体上。
第5项：根据第4项所述的方法，其中gag、pol基因的序列位于一个构建体上，rev基因的序列位于另一个构建体上，病毒包膜蛋白的编码序列位于第三个构建体上，并且携带目的核酸片段的病毒基因组转录盒序列位于第四个构建体上。
第6项：根据第2项所述的方法，其中所述慢病毒的gag、pol和rev基因是HIV-1病毒的gag、pol和rev基因。
第7项：根据第2项所述的方法，其中所述病毒包膜蛋白选自猫白血病病毒(RD114)包膜蛋白、两性逆转录病毒包膜蛋白、嗜性逆转录病毒包膜蛋白、狒狒猿白血病病毒包膜蛋白、尼帕病毒包膜蛋白、莫科拉病毒包膜蛋白、淋巴细胞脉络膜脑膜炎病毒包膜蛋白、基孔肯雅病毒包膜蛋白、罗斯河病毒包膜蛋白、塞姆利基森林病毒包膜蛋白、信德比斯病毒包膜蛋白、委内瑞拉马脑炎病毒包膜蛋白、西部马脑炎病毒包膜蛋白、流感病毒包膜蛋白、禽瘟病毒包膜蛋白、昌迪普拉病毒和皮里病毒包膜蛋白、猿免疫缺陷病毒包膜蛋白、猫免疫缺陷病毒包膜蛋白、马传染性贫血病毒包膜蛋白、埃博拉病毒包膜蛋白、狂犬病病毒包膜蛋白、杆状病毒包膜蛋白、丙型肝炎病毒包膜蛋白、猫内源反转录病毒包膜蛋白、麻疹病毒包膜蛋白、鼠白血病病毒的兼嗜性4070A和10A1、长臂猿白血病病毒的包膜蛋白、人免疫缺陷病毒的gp120和水疱性口炎病毒的糖蛋白(VSV-G)。
第8项：根据第2项所述的方法，其中所述病毒包膜蛋白是水疱性口炎病毒的糖蛋白(VSV-G)。
第9项：根据第2项所述的方法，其中所述SB系统中使用的转座酶是SB100X，和/或所述PB系统中使用的转座酶是ePiggyBac。
第10项：根据第2项所述的方法，其中所述gag、pol和rev基因、病毒包膜蛋白的编码序列和携带目的核酸片段的病毒基因组转录盒中的一种或多种的转录是受控的。
第11项：根据第10项所述的方法，其中所述病毒包膜蛋白是VSV-G并且rev基因和VSV-G的编码序列的转录是受控的。
第12项：根据第10项所述的方法，其中所述病毒包膜蛋白是VSV-G并且gag、pol和rev基因以及VSV-G的编码序列的转录是受控的。
第13项：根据第10项所述的方法，其中所述受控的转录是通过将所述基因或序列置于诱导表达系统的控制下实现的。
第14项：根据第13项所述的方法，其中所述诱导表达系统选自四环素诱导表达系统和Cumate诱导表达系统。
第15项：根据第14项所述的方法，其中所述基因或序列被置于Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下。
第16项：根据第15项所述的方法，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，和/或VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列、TRE 3GCuO序列或TRE 3GCuO序列-内含子的控制下，和/或VSV-G的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子、TRE adv序列-内含子、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下。
第17项：根据第16项所述的方法，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列或TRE 3GCuO序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3GCuO序列-内含子的控制下。
第18项：根据第17项所述的方法，其中在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在TRE 3GCuO序列-内含子的控制下，并且gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为2-8个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为1:1至4:1。
第19项：根据第18项所述的方法，其中gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为4-6个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为2:1至3:1。
第20项：根据第15项所述的方法，其中利用SB转座子系统或PB转座子系统将Tet-On反式激活物蛋白的编码序列或Tet-On反式激活物蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列整合在所述宿主细胞的基因组中。
第21项：根据第20项所述的方法，其中所述Tet-On反式激活物蛋白是rtTA 3G。
第22项：根据第15项所述的方法，其中利用SB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列以及Tet-On反式激活物蛋白的编码序列或Tet-On反式激活物蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用PB 转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列以及Tet-On反式激活物蛋白的编码序列或Tet-On反式激活物蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用SB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中。
第23项：通过根据第1-22项中任一项所述的方法制备的生产细胞。
第24项：根据第23项所述的生产细胞，其中所述生产细胞于2020年4月13日以保藏编号CGMCC No.19675保藏于中国普通微生物菌种保藏管理中心。
第25项：一种用于生产携带目的核酸片段的逆转录病毒载体的生产细胞，所述生产细胞在其基因组中整合有逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列，所述gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列的两端分别具有用于被SB转座酶识别的IR/DR序列或用于被PB转座酶识别的ITR序列，并且所述IR/DR序列和所述ITR序列在所述生产细胞中同时存在。
第26项：根据第25项所述的生产细胞，其中所述gag和pol基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被SB转座酶识别的IR/DR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被PB转座酶识别的ITR序列，或者所述gag和pol基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被PB转座酶识别的ITR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被SB转座酶识别的IR/DR序列。
第27项：根据第25项所述的生产细胞，其中所述逆转录病毒是慢病毒，所述生产细胞在其基因组中还整合有慢病毒的rev基因的序列，并且所述rev基因的序列的两端具有用于被SB转座酶识别的IR/DR序列或用于被PB转座酶识别的ITR序列。
第28项：根据第27项所述的生产细胞，其中所述逆转录病毒是HIV-1病毒。
第29项：根据第27项所述的生产细胞，其中所述gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被SB转座酶识别的IR/DR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被PB转座酶识别的ITR序列，或者所述gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被PB转座酶识别的ITR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被SB转座酶识别的IR/DR序列。
第30项：根据第25项所述的生产细胞，其中所述病毒包膜蛋白是水疱性口炎病毒的糖蛋白(VSV-G)。
第31项：根据第23项所述的生产细胞或根据第25项所述的生产细胞，其中所述目的核酸片段序列两端具有用于位点特异性重组酶系统的识别序列，和/或所述外包膜蛋白的编码序列两端具有用于位点特异性重组酶系统的识别序列。
第32项：用于逆转录病毒载体的目的核酸片段和/或外包膜蛋白替换系统，所述系统包括：根据第31项所述的生产细胞，位点特异性重组酶系统，以及待替换的目的核酸片段和/或外包膜蛋白的编码序列。
第33项：根据第32项所述的系统，其中所述位点特异性重组酶系统是FLP-FRT或Cre-lox重组酶系统。
第34项：根据第32项所述的系统，其中所述生产细胞中原有的目的核酸片段包含标记基因或与标记基因相连和/或所述生产细胞中原有的外包膜蛋白的编码序列与标记基因相连，并且与待替换的目的核酸片段和/或外包膜蛋白的编码序列相连的标记基因与所述生产细胞中原有的标记基因不同。
第35项：根据第34项所述的系统，其中所述标记基因是用于真核细胞的抗性基因序列，代谢通路筛选基因，荧光蛋白标记基因，能够用于报告基因检测蛋白酶基因，或其任意组合。
第36项：根据第35项所述的系统，其中所述抗性基因序列选自：潮霉素抗性基因、嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、博莱霉素抗性基因。
第37项：根据第35项所述的系统，其中所述代谢通路筛选基因选自编码二氢叶酸还原酶(Dihydrofolate reductase)、谷氨酰胺合成酶(Glutamine synthetase)、胸苷激酶(Thymidine kinase)的基因。
第38项：根据第35项所述的系统，其中所述荧光蛋白标记基因选自编码绿色荧光蛋白(EGFP)、红色荧光蛋白(dsRed)、樱桃色荧光蛋白(mcherry)、青色荧光蛋白(ECFP)、黄色荧光蛋白(EYFP)、以及荧光蛋白的其它突变衍生蛋白的基因序列。
第39项：根据第35项所述的系统，其中所述能够用于报告基因检测蛋白酶基因选自编码萤光素酶(luciferase)、β-半乳糖苷酶(β-galactosidase)、氯霉素乙酰转移酶(chloramphenical acetyltransferase)的基因序列。
第40项：用于替换逆转录病毒的生产细胞中的目的核酸片段和/或外包膜蛋白的方法，所述方法包括：提供根据第31项所述的生产细胞，以及利用位点特异性重组酶系统将生产细胞中原有的目的核酸片段和/或外包膜蛋白的编码序列替换为待替换的目的核酸片段和/或外包膜蛋白的编码序列。
第41项：根据第40项所述的方法，其中所述位点特异性重组酶系统是FLP-FRT或Cre-lox重组酶系统。
第42项：根据第40项所述的方法，其中所述生产细胞中原有的目的核酸片段包含标记基因或与标记基因相连和/或所述生产细胞中原有的外包膜蛋白的编码序列与标记基因相连，并且与待替换的目的核酸片段和/或外包膜蛋白的编码序列相连的标记基因与所述生产细胞中原有的标记基因不同。
第43项：根据第42项所述的方法，其中所述标记基因是用于真核细胞的抗性基因序列，代谢通路筛选基因，荧光蛋白标记基因，能够用于报告基因检测蛋白酶基因，或其任意组合。
第44项：根据第43项所述的方法，其中所述抗性基因序列选自：潮霉素抗性基因、嘌呤霉素抗性 基因、新霉素抗性基因、杀稻瘟素抗性基因、博莱霉素抗性基因。
第45项：根据第43项所述的方法，其中所述代谢通路筛选基因选自编码二氢叶酸还原酶(Dihydrofolate reductase)、谷氨酰胺合成酶(Glutamine synthetase)、胸苷激酶(Thymidine kinase)的基因。
第46项：根据第43项所述的方法，其中所述荧光蛋白标记基因选自编码绿色荧光蛋白(EGFP)、红色荧光蛋白(dsRed)、樱桃色荧光蛋白(mcherry)、青色荧光蛋白(ECFP)、黄色荧光蛋白(EYFP)、以及荧光蛋白的其它突变衍生蛋白的基因序列。
第47项：根据第43项所述的方法，其中所述能够用于报告基因检测蛋白酶基因选自编码萤光素酶(luciferase)、β-半乳糖苷酶(β-galactosidase)、氯霉素乙酰转移酶(chloramphenical acetyltransferase)的基因序列。
第48项：通过根据第40项所述的用于替换逆转录病毒的生产细胞中的目的核酸片段和/或外包膜蛋白的方法获得的逆转录病毒的生产细胞。
第49项：一种用于生产携带目的核酸片段的逆转录病毒载体的方法，所述方法包括以下步骤：
向根据第23-31项中任一项所述的生产细胞或根据第48项所述的生产细胞加入诱导表达系统的诱导物，和
收集和纯化所得的携带目的核酸片段的逆转录病毒载体。
第50项：根据第49项所述的方法，其中所述诱导物是四环素或四环素衍生物和/或Cumate及其功能类似物。
第51项：根据第50项所述的方法，其中所述四环素衍生物是多西环素。
第52项：通过根据第49项所述的方法制备的携带目的核酸片段的逆转录病毒载体。
第53项：根据第23-31项中任一项所述的生产细胞、根据第48项所述的生产细胞或根据第52项所述的携带目的核酸片段的逆转录病毒载体在制备用于向细胞递送目的核酸片段的试剂中的用途。
第54项：一种用于制备用于生产慢病毒载体的包装/生产细胞的方法，所述方法包括将慢病毒的gag、pol和rev基因的序列以及水疱性口炎病毒的糖蛋白(VSV-G)的编码序列转入宿主细胞中，所述gag、pol和rev基因以及VSV-G的编码序列中的一种或多种的转录在Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，和/或VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列、TRE 3GCuO序列或TRE 3GCuO序列-内含子的控制下，和/或VSV-G的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子、TRE adv序列-内含子、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下。
第55项：根据第54项所述的方法，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列或TRE 3GCuO序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3GCuO序列-内含子的控制下。
第56项：根据第54项所述的方法，其中所述慢病毒的gag、pol和rev基因的序列以及VSV-G的编码序列被整合在宿主细胞的基因组中。
第57项：根据第56项所述的方法，其中通过SB转座子系统或PB转座子系统将慢病毒的gag、pol和rev基因的序列以及VSV-G的编码序列整合在宿主细胞的基因组中。
第58项：根据第56项所述的方法，其中在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在TRE 3GCuO序列-内含子的控制下，并且gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为2-8个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为1:1至4:1。
第59项：根据第58项所述的方法，其中gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为4-6个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为2:1至3:1。
第60项：根据第56项所述的方法，其中在通过SB转座子系统或PB转座子系统将慢病毒的gag、pol和rev基因的序列以及VSV-G的编码序列整合在宿主细胞的基因组中后，通过相同的转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中，或通过瞬时转染的方法将携带目的核酸片段的病毒基因组转录盒序列进一步转入所述宿主细胞中。
第61项：一种用于生产慢病毒载体的包装/生产细胞，所述包装/生产细胞包含慢病毒的gag、pol和rev基因的序列以及水疱性口炎病毒的糖蛋白(VSV-G)的编码序列，所述gag、pol和rev基因以及VSV-G的编码序列中的一种或多种的转录在Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，和/或VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列、TRE 3GCuO序列或TRE 3GCuO序列-内含子的控制下，和/或VSV-G的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子、TRE adv序列-内含子、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下。
第62项：根据第61项所述的用于生产慢病毒载体的包装/生产细胞，其中所述慢病毒的gag基因的序列及/或pol基因的序列及/或rev基因的序列及/或水疱性口炎病毒的糖蛋白(VSV-G)的编码序列被整合 在所述包装/生产细胞的基因组中。
第63项：根据第61项所述的用于生产慢病毒载体的包装/生产细胞，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列或TRE 3GCuO序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3GCuO序列-内含子的控制下。
第64项：根据第61项所述的用于生产慢病毒载体的包装/生产细胞，其中在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在TRE 3GCuO序列-内含子的控制下，并且gag/pol基因在所述包装/生产细胞的基因组中的插入拷贝数为2-8个拷贝/细胞，并且插入在所述包装/生产细胞的基因组中的gag/pol与VSV-G的拷贝数之比为1:1至4:1。
第65项：根据第64项所述的用于生产慢病毒载体的包装/生产细胞，其中gag/pol基因在所述包装/生产细胞的基因组中的插入拷贝数为4-6个拷贝/细胞，并且插入在所述包装/生产细胞的基因组中的gag/pol与VSV-G的拷贝数之比为2:1至3:1。
第66项：根据第61项所述的用于生产慢病毒载体的包装/生产细胞，其于2020年4月13日以保藏编号CGMCC No.19674保藏于中国普通微生物菌种保藏管理中心。
第67项：一种生产慢病毒载体的方法，其包括在适于生产慢病毒载体的条件下培养根据第61至66任一项项所述的包装/生产细胞。
附图说明
图1示实施例所用部分质粒的图谱。
图2示实施例2中验证SB和PB转座子系统的转座专一性。横坐标为细胞传代代次，纵坐标为各共转染组合细胞EGFP阳性比例(图2A)和EGFP荧光强度中位值(MFI)(图2B)。
图3示实施例2中构建稳定生产慢病毒载体的生产细胞的实验流程图。
图4示实施例2中通过SB和PB转座子系统按照不同组合方式构建的慢病毒稳定生产细胞的产毒能力检测结果，其中横坐标为构建的生产细胞编号，纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。
图5示实施例3中对比不同调控序列调控单一慢病毒包装基因对产毒能力的影响。图示为病毒滴度检测结果，横坐标为待测的不同调控序列名称；纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。rev(图5A)、VSV-G(图5B)和gag/pol(图5C)在不同调控序列调控下的诱导产毒滴度和无诱导泄露滴度的检测结果。
图6示实施例3中使用优化后的不同调控序列组合调控rev、VSV-G和gag/pol的表达并对比诱导产毒滴度和无诱导泄露滴度。图示为病毒滴度检测结果。横坐标为具有不同调控序列的质粒组合，纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。
图7示实施例4中单基因在宿主细胞基因组整合的拷贝数对细胞产毒能力的影响。横坐标表示转染细胞时不同质粒的摩尔比例，左侧纵坐标为检测病毒转染滴度的Luciferase实验的RLU值，右侧纵坐标为待测基因在细胞基因组的平均整合拷贝数。图7A示rtTA 3G-CymR片段基因组整合拷贝数对产毒能力的影响，图7B示rev片段基因组整合拷贝数对产毒能力的影响，图7C示VSV-G片段基因组整合拷贝数对产毒能力的影响，图7D示gag/pol片段基因组整合拷贝数对产毒能力的影响。
图8示实施例4中gag/pol和VSV-G片段在宿主细胞基因组中不同的整合拷贝数比例对细胞产毒能力的影响。横坐标表示转染细胞时不同质粒的摩尔比例，左侧纵坐标为检测病毒转染滴度的Luciferase实验的RLU值，右侧纵坐标为gag/pol和VSV-G片段在细胞基因组中整合拷贝数的比值。
图9示实施例5中EuLV293T3rd慢病毒稳定包装细胞系构建、筛选及悬浮适应流程图。
图10示实施例5中SB16单克隆(图10A)和SB28单克隆(图10B)筛选过程及96孔板、24孔板和6孔板筛选结果。横坐标为单克隆细胞制备病毒与Luciferase标准品RLU检测结果的比值(表示为以2为底的指数)，纵坐标为在各个数据区间内单克隆细胞数目。图中····虚线为所有单克隆样品RLU比值的平均值，方框为根据检测结果筛选保留的单克隆细胞株。
图11示实施例5中使用HT1080细胞Luciferase病毒滴度检测方法检测适应悬浮培养的单克隆包装细胞株产毒能力的检测结果。其中横坐标为检测的细胞编号，纵坐标为与病毒转染滴度相应的Luciferase检测实验的RLU值。
图12示实施例6中瞬时转染不同慢病毒包装基因质粒组合对贴壁培养的高产单克隆包装细胞株的产毒滴度影响。横坐标图例表示瞬时转染的慢病毒包装基因组合，其中“-”号代表无诱导条件，“+”代表诱导条件；纵坐标为病毒转染滴度对应的Luciferase检测实验的RLU值；实线代表通过瞬时转染293T细胞阳性对照的产毒滴度；虚线代表各细胞诱导产毒阳性对照的产毒滴度。
图13示实施例6中瞬时转染不同慢病毒包装基因质粒组合对悬浮培养的高产单克隆包装细胞株的产毒滴度影响。横坐标图例表示瞬时转染的慢病毒包装基因组合，其中“-”号代表无诱导条件，“+”代表诱导条件；纵坐标为病毒转染滴度对应的Luciferase检测实验的RLU值；实线代表通过瞬时转染293T细胞阳性对照的产毒滴度；虚线代表各细胞诱导产毒阳性对照的产毒滴度。
图14示实施例7中构建的携带不同目的核酸片段的慢病毒稳定生产细胞系的产毒能力和瞬时转染方法产毒能力对比。横坐标图例表示检测的转移载体质粒编号；纵坐标为病毒转染滴度对应的Luciferase检测实验的RLU值
图15示实施例7中构建的慢病毒稳定生产细胞在无血清悬浮培养条件下的诱导及泄露产毒滴度。 横坐标图例“-”号代表无诱导条件，“+”代表诱导条件；纵坐标为病毒转染滴度对应的Luciferase检测实验的RLU值。
本发明的实施方式
提供以下实施例用以对本发明的技术方案进行说明，以下实施例不应被认为是对本发明的范围和精神的限制。
实施例1：质粒构建方法
以下实施例所使用的分子克隆技术，例如，DNA片段的PCR扩增、DNA片段的限制性内切酶酶切、DNA片段的凝胶回收、两段或多段DNA片段的T4 DNA连接酶连接、连接产物感受态细胞的转化、质粒小量制备及鉴定等方法均为本领域熟知技术。以下实施例中涉及以下试剂：PCR酶(Thermo，F-530S)；限制性内切酶(NEB)；T4 DNA连接酶(Invitrogen，15224041)；DNA片段凝胶回收试剂盒(Omega，D2500-02)；质粒小提试剂盒(TIANGEN，DP105-03)；感受态细胞(XL-10 Gold，湖南丰晖生物科技有限公司，JZ011)；SEQ ID NO:1至SEQ ID NO:18所示的核酸序列由金斯瑞合成并用于本公开所述质粒构建，质粒测序鉴定由Invitrogen公司完成。以下实施例所用部分质粒的图谱简图如图1所示；表1为构建质粒的引物信息；表2为序列SEQ ID NO:1至SEQ ID NO：31的元件组成说明；表3为质粒中各功能元件说明；表4为本公开构建的质粒编号及对应名称。以下实施例中所涉及的各质粒所采用的功能元件序列信息为实现本公开的示例，本领域技术人员可以预期将以下实施例中所用质粒上各功能元件序列替换成其它生物学功能类似的元件序列也能达到本公开所述效果，包括但不限于质粒的骨架序列(如复制原点(replication origin)、抗性基因等)、酶切位点序列、转座子重复序列、诱导系统响应元件序列、隔离子(Insulator)序列、启动子序列、内含子序列、聚腺苷酸信号(PolyA)序列、不同密码子优化的基因序列、以上各功能元件序列和基因序列的突变体、以及各功能元件序列和基因序列的克隆位置、克隆顺序和克隆方向。具体的质粒构建方法如下所示：
1.构建质粒18BF007和18BF004：将合成序列SEQ ID NO:2(2900bp)和序列SEQ ID NO:3(1386bp)用NotI和AsiSI酶切并分别连接在质粒18BF003(序列SEQ ID NO:1，1893bp)的NotI和AsiSI酶切位点，从而分别构建得到质粒18BF007和18BF004。
2.构建质粒18BF011和18BF063：将18BF007质粒用MluI和SphI酶切，凝胶回收1730bp片段并将其连接在18BF003质粒的MluI和SphI酶切位点从而构建得到质粒18BF011。将合成序列SEQ ID NO:4(915bp)用MluI和ClaI酶切并连接在18BF007的MluI和ClaI酶切位点，替换CMV启动子构建质粒18BF063。
3.构建质粒18BF072、18BF071、19BF249、19BF248、19BF247、19BF246、18BF070和18BF069：以pRSV-Rev(Addgene，#12253)为模板、C-rev-F(SEQ ID NO:34)和C-rev-R(SEQ ID NO:35)为引物PCR 扩增rev基因片段(380bp)，之后用ClaI和XhoI酶切并连接在18BF063质粒的ClaI和XhoI酶切位点构建18BF072质粒。将合成序列SEQ ID NO:5(887bp)、序列SEQ ID NO:6(897bp)和序列SEQ ID NO:7(852bp)用MluI和ClaI酶切并连接在18BF072质粒的MluI和ClaI酶切位点，替换TRE 3GCuO-BGI片段从而分别构建得到质粒18BF071、19BF249和19BF248。将质粒18BF072、18BF071、19BF249和19BF248用BstBI酶切，分别凝胶回收4147bp(18BF072)，4119bp(18BF071)，4129bp(19BF249)和4084bp(19BF248)片段并用T4连接酶连接以分别构建质粒19BF247、19BF246、18BF070和18BF069。
4.构建质粒18BF068、18BF067、19BF245、19BF244、19BF243、19BF242、18BF066、18BF065和19BF254：以pMD2.G(Addgene，#12259)为模板，C-VSVG-F(SEQ ID NO:32)和C-VSVG-R(SEQ ID NO:33)为引物PCR扩增VSV-G基因片段(1565bp)，之后用ClaI和XhoI酶切并连接在18BF063质粒的ClaI和XhoI酶切位点以构建得到质粒18BF068。将合成序列SEQ ID NO:5(887bp)、序列SEQ ID NO:6(897bp)和序列SEQ ID NO:7(852bp)分别用MluI和ClaI酶切并连接在18BF068质粒的MluI和ClaI酶切位点，替换TRE 3GCuO-BGI片段从而构建得到质粒18BF067、19BF245和19BF244。将质粒18BF068用ClaI和XhoI酶切，凝胶回收1550bp片段并分别将其连接在质粒19BF247、19BF246、18BF070和18BF069的ClaI和XhoI酶切位点，替换其中rev基因从而分别构建得到质粒19BF243、19BF242、18BF066和18BF065。将合成序列SEQ ID NO:8(887bp)用SpeI和PvuII酶切并连接在18BF068质粒的AvrII和PmeI酶切位点从而构建得到质粒19BF254。
5.构建质粒18BF074、19BF131、19BF130、19BF251、19BF250、19BF126、19BF129，19BF128和18BF076：以pMDLg/pRRE(Addgene，#12251)为模板、C-RRE-F(SEQ ID NO:36)和C-RRE-R(SEQ ID NO:37)为引物PCR扩增RRE片段(400bp)；C-GagPol-F(SEQ ID NO:38)和C-GagPol-R(SEQ ID NO:39)为引物PCR扩增gag/pol基因片段(4336bp)，之后用XbaI和XhoI以及EcoRI和XbaI分别酶切处理两个DNA片段并连接在18BF007质粒的EcoRI和XhoI酶切位点从而构建得到18BF074质粒。将合成序列SEQ ID NO:4(915bp)、序列SEQ ID NO:5(887bp)、序列SEQ ID NO:6(897bp)和序列SEQ ID NO:7(852bp)分别用MluI和EcoRI酶切并连接在18BF074质粒的MluI和EcoRI酶切位点，替换CMV-BGI片段从而构建得到19BF131、19BF130、19BF251和19BF250质粒。将18BF074、19BF131、19BF130和19BF250质粒分别用BstBI酶切，分别凝胶回收8758bp(18BF074)，8494bp(18BF131)，8466bp(19BF130)和8421bp(19BF250)片段并用T4连接酶连接从而分别构建得到质粒19BF126、19BF129、19BF128和18BF076。
6.构建质粒19BF257、19BF256、19BF075和19BF074：将合成序列SEQ ID NO:9(633bp)和序列SEQ ID NO:10(1496bp)分别用ClaI和XhoI以及SpeI和AgeI酶切处理并依次连接在18BF007质粒的ClaI和XhoI酶切位点以及AvrII和AgeI酶切位点从而构建得到19BF073质粒。将合成序列SEQ ID NO:11(1979bp)用MluI和AgeI酶切并连接在18BF007质粒的MluI和AgeI酶切位点，替换CMV-BGI-MCS-pA片段，从而构建得到18BF008质粒。将合成序列SEQ ID NO:12(768bp)和序列SEQ ID NO:13(765bp)分 别用ClaI和XhoI酶切并分别连接在18BF008质粒的ClaI和XhoI酶切位点从而分别构建得到18BF085和18BF084质粒。将合成序列SEQ ID NO:10(1496bp)用SpeI和AgeI酶切并分别连接在18BF085和18BF084质粒的AvrII和AgeI酶切位点从而分别构建得到19BF257和19BF256质粒。将质粒19BF073用SpeI和AgeI酶切，凝胶回收3821bp片段并将其分别连接在18BF085和18BF084质粒的AvrII和AgeI酶切位点从而分别构建得到19BF075和19BF074质粒。
7.构建质粒18BF019和18BF031：将合成序列SEQ ID NO:15(1044bp)和序列SEQ ID NO:14(1320bp)分别用BamHI和XhoI以及XhoI和BglII酶切并连接在18BF011质粒的BamHI和BglII酶切位点从而构建得到18BF019质粒。将合成序列SEQ ID NO:16(1806bp)和序列SEQ ID NO:14(1320bp)分别用BamHI和XhoI以及XhoI和BglII酶切并连接在18BF011质粒的BamHI和BglII酶切位点从而构建得到18BF031质粒。
8.构建质粒18BF094、19BF255、18BF091、18BF096和19BF252：将质粒18BF071和19BF254分别用PacI和AvrII酶切，分别凝胶回收DNA片段1762bp和3838bp，并将回收的两种片段分别连接在18BF004质粒的PacI和AvrII酶切位点从而分别构建得到18BF094和19BF255质粒。将质粒18BF068用PacI、AvrII和PvuI酶切，凝胶回收DNA片段2975bp，并将回收的DNA片段连接在18BF004质粒的PacI和AvrII酶切位点从而构建得到18BF091质粒。将质粒18BF074用PacI和PmeI酶切，凝胶回收DNA片段6428bp，并将其连接在18BF004质粒的PacI和PmeI酶切位点从而构建得到18BF096质粒。将质粒19BF074用SpeI和PmeI酶切，凝胶回收DNA片段6513bp，并将其连接在18BF004质粒的SpeI和PmeI酶切位点从而构建得到19BF252质粒。
9.构建质粒19BF081、19BF217和19BF218：以pRRLSIN.cPPT.PGK-GFP.WPRE(Addgene，#12252)为模板，hPGK-F(SEQ ID NO:40)和hPGK-R(SEQ ID NO:41)为引物PCR扩增PGK基因片段(706bp)；以pGL3-Basic(Promega，E1751)为模板，Luc-F(SEQ ID NO:42)和Luc-R(SEQ ID NO:43)为引物PCR扩增luciferase基因片段(1728bp)，之后用MluI和BamHI(凝胶回收538bp片段)以及BamHI和XhoI分别酶切处理两个DNA片段并连接在19BF126质粒的MluI和XhoI酶切位点，替换原质粒DNA片段从而构建得到18YYH26质粒。用TRIzol(ThermoFisher 15596026)试剂抽提纯化HepG2(ATCC HB-8065)细胞和健康人外周血单核细胞(PBMC)的总RNA；依照SuperScript IV(ThermoFisher 18090010)说明并以HepG2和PBMC混合总RNA做为模板制作cDNA库；并以此cDNA库为模板，F8V1-F(SEQ ID NO:44)和F8V1HA-R(SEQ ID NO:45)为引物PCR扩增F8cHA基因片段(PCR产物长度7116bp，经DNA测序PCR产物18bp至7070bp序列和NM_000132.3序列中172bp至7224bp序列一致)；之后用ClaI和XhoI酶切处理并连接在19BF126质粒的ClaI和XhoI酶切位点，替换原质粒DNA片段从而构建得到19BF215质粒。以19BF215质粒为模板，分别以F8V1F(SEQ ID NO:44)和FP-BDDF8-R(SEQ ID NO:47)以及FP-BDDF8-F(SEQ ID NO:46)和F8V1HA-R(SEQ ID NO:45)为引物PCR扩增两段DNA片段2317bp和 2154bp；用融合PCR方法将这两段DNA连接并以F8V1F(SEQ ID NO:44)和F8V1HA-R(SEQ ID NO:45)为引物PCR扩增得到BDDF8cHA(4434bp)基因片段；之后用ClaI和XhoI酶切处理并连接在19BF126质粒的ClaI和XhoI酶切位点，替换原质粒DNA片段从而构建得到19BF216质粒。将合成序列SEQ ID NO:18(3610bp)用SpeI和AgeI酶切并连接在18BF004质粒的SpeI和AgeI酶切位点从而构建得到19BF080质粒。将合成序列SEQ ID NO:17(1320bp)用XhoI和BglII酶切并连接在19BF080质粒的Xho I和BamH I酶切位点从而构建得到19BF214质粒。将质粒18YYH26、19BF215和19BF216分别用Pac I和Xho I酶切并分别凝胶回收DNA片段2272bp、7792bp和5110bp，并将回收的片段分别连接在19BF214质粒的Pac I和Xho I酶切位点从而分别构建得到19BF081、19BF217和19BF218质粒。
10.构建质粒18BF022、18BF033和19BF078：将合成序列SEQ ID NO:17(1320bp)用Xho I和Bgl II酶切并连接在18BF007质粒的Xho I和Bgl II酶切位点从而构建得到18BF022质粒。将质粒18BF022用Mlu I、Age I和Pvu I酶切，凝胶回收DNA片段3052bp，并将其连接在18BF004质粒的Mlu I和Age I酶切位点从而构建得到18BF033质粒。将合成序列SEQ ID NO:18(3610bp)用Spe I和Age I酶切并连接在18BF007质粒的Spe I和Age I酶切位点，替换原质粒DNA片段从而构建得到19BF077质粒。将质粒19BF081用Pac I和Nde I酶切，凝胶回收DNA片段3598bp，并将其连接在19BF077质粒的Pac I和Nde I酶切位点从而构建得到19BF078质粒。
表1.引物信息列表
Figure PCTCN2020115522-appb-000002
表2.序列元件组成说明
Figure PCTCN2020115522-appb-000003
Figure PCTCN2020115522-appb-000004
表3.质粒功能元件说明
Figure PCTCN2020115522-appb-000005
Figure PCTCN2020115522-appb-000006
表4.质粒编号及名称
质粒编号 质粒名称
18BF003 pma-MCS
18BF007 pmaSBT3-2xHS4I-CMV-BGI-MCS
18BF004 pmaHPBT-2xHS4I-MCS
18BF011 pmaCMV-BGI-MCS
18BF063 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-MCS
18BF072 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-rev
18BF071 pmaSBT3-2xHS4I-TRE 3G-BGI-rev
19BF249 pmaSBT3-2xHS4I-TRE advCuO-BGI-rev
19BF248 pmaSBT3-2xHS4I-TRE adv-BGI-rev
19BF247 pmaSBT3-2xHS4I-TRE 3GCuO-rev
19BF246 pmaSBT3-2xHS4I-TRE 3G-rev
18BF070 pmaSBT3-2xHS4I-TRE advCuO-rev
18BF069 pmaSBT3-2xHS4I-TRE adv-rev
18BF068 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-VSVG
18BF067 pmaSBT3-2xHS4I-TRE 3G-BGI-VSVG
19BF245 pmaSBT3-2xHS4I-TRE advCuO-BGI-VSVG
19BF244 pmaSBT3-2xHS4I-TRE adv-BGI-VSVG
19BF243 pmaSBT3-2xHS4I-TRE 3GCuO-VSVG
19BF242 pmaSBT3-2xHS4I-TRE 3G-VSVG
18BF066 pmaSBT3-2xHS4I-TRE advCuO-VSVG
18BF065 pmaSBT3-2xHS4I-TRE adv-VSVG
19BF254 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-VSVG-optiBSD
18BF074 pmaSBT3-2xHS4I-CMV-BGI-gag/pol-RRE
19BF131 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-gag/pol-RRE
19BF130 pmaSBT3-2xHS4I-TRE 3G-BGI-gag/pol-RRE
19BF251 pmaSBT3-2xHS4I-TRE advCuO-BGI-gag/pol-RRE
19BF250 pmaSBT3-2xHS4I-TRE adv-BGI-gag/pol-RRE
19BF126 pmaSBT3-2xHS4I-CMV-gag/pol-RRE
19BF129 pmaSBT3-2xHS4I-TRE 3GCuO-gag/pol-RRE
19BF128 pmaSBT3-2xHS4I-TRE 3G-gag/pol-RRE
18BF076 pmaSBT3-2xHS4I-TRE adv-gag/pol-RRE
19BF073 pmaSBT3-2xHS4I-CMV-BGI-optiCymR-optiHygroR
18BF008 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-MCS
18BF085 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G
18BF084 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv
19BF257 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G-optiHygroR
19BF256 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv-optiHygroR
19BF075 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G-CMV-BGI-optiCymR-optiHygroR
19BF074 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv-CMV-BGI-optiCymR-optiHygroR
18BF019 pmaCMV-BGI-optiSB-IRES-ECFP
18BF031 pmaCMV-BGI-optiPB-IRES-ECFP
18BF094 pmaHPBT-2xHS4I-TRE 3G-BGI-rev
19BF255 pmaHPBT-2xHS4I-TRE 3GCuO-BGI-VSVG-optiBSD
18BF091 pmaHPBT-2xHS4I-TRE 3GCuO-BGI-VSVG
18BF096 pmaHPBT-2xHS4I-CMV-BGI-gag/pol-RRE
19BF252 pmaHPBT-2xHS4I-CAGGS-BGI(C&R)-rtTA adv-CMV-BGI-optiCymR-optiHygroR
18YYH26 pmaSBT3-2xHS4I-hPGK-Luc
19BF215 pmaSBT3-2xHS4I-CMV-F8cHA
19BF216 pmaSBT3-2xHS4I-CMV-BDDF8cHA
19BF080 pmaHPBT-2xHS4I-LVRSV-MCS-optiPuroR
19BF214 pmaHPBT-2xHS4I-LVRSV-MCS-IRES-EGFP-optiPuroR
19BF081 pmaHPBT-2xHS4I-LVRSV-hPGK-Luc-IRES-EGFP-optiPuroR
19BF217 pmaHPBT-2xHS4I-LVRSV-CMV-F8cHA-IRES-EGFP-PuroR
19BF218 pmaHPBT-2xHS4I-LVRSV-CMV-BDDF8cHA-IRES-EGFP-optiPuroR
18BF022 pmaSBT3-2xHS4I-CMV-BGI-MCS-IRES-EGFP
18BF033 pmaHPBT-2xHS4I-CMV-BGI-MCS-IRES-EGFP
19BF077 pmaSBT3-2xHS4I-LVRSV-MCS-optiPuroR
19BF078 pmaSTB3-2xHS4I-LVRSV-hPGK-Luc-IRES-EGFP-optiPuroR
实施例2：使用SB和PB双转座子系统构建慢病毒生产细胞系
1.验证SB和PB转座子系统专一性
使用SB和PB双转座子系统构建慢病毒生产细胞系前需证明SB和PB转座子系统的专一性，即SB和PB转座酶是否仅能识别各自的转座子结合序列。专一性验证实验通过在293T细胞中分别转染携带EGFP的SB/PB转座子质粒(18BF022/18BF033)和SB/PB转座酶质粒(18BF019/18BF031)，然后检测在连续传代中293T细胞中EGFP信号丢失率来评定两套转座子系统是否能交叉工作，具体步骤如下：
将293T细胞(ATCC，CRL3216)培养在37℃、5％CO 2环境下，培养基为DMEM完全培养基(DMEM(Sigam，D6429)添加10％FBS(ExCell，11H116)。将293T细胞按8E5个细胞每孔接种于6孔板(Corning，3516)中。培养24小时后，按照《MolecμLar Cloning:A Laboratory Manual(Fourth edition)Chapter15，Michael R.Green，Cold Spring Harbor Laboratory Press，2012》中磷酸钙转染方式配制转染试剂，每孔加入200μL转染试剂，其中含有0.12mol/L氯化钙，1xHEPES缓冲液，5.5μg总质粒量。其中SB和PB的转座子质粒分别为18BF022和18BF033，报告基因都为EGFP。SB和PB转座酶质粒分别为18BF019和18BF031。转座子质粒和转座酶质粒按照摩尔质量10:1进行磷酸钙共转染。实验共设置6个共转染组合，分别为(1)SB转座子和SB转座酶(18BF022+18BF019)；(2)PB转座子和PB转座酶 (18BF033+18BF031)；(3)SB转座子和PB转座酶(18BF022+18BF031)；(4)PB转座子和SB转座酶(18BF033+18BF019)；(5)SB转座子和空质粒(18BF022+18BF003)；(6)PB转座子和空质粒(18BF033+18BF003)。转染6小时后，更换培养基为新的DMEM完全培养基。转染24小时后，用胰酶(Sigam，T4799)消化细胞并使用流式细胞仪(ACEA，NovoCyte3130)检测细胞EGFP阳性比例和荧光强度中位值(MFI)，此数据记为P1代数据。之后在无筛选压力下培养细胞并维持每三天一次传代操作，每次传代用流式法检测EGFP的阳性比例和MFI值，一共连续记录5个代次。
图2为SB和PB专一性验证实验结果，横坐标为细胞传代代次，纵坐标为各共转染组合细胞EGFP阳性比例(图2A)和荧光强度中位值(MFI)(图2B)。如图所示：6组样品在转染后P1代的EGFP阳性比例在61.1％-79.1％之间，MFI在32685-44827之间。之后在持续无筛选压力培养过程中，EGFP阳性比例快速下降，但SB实验组18BF022+18BF019和PB实验组18BF033+18BF031在P2代之后EGFP下降明显减缓并在P5代时EGFP阳性比例稳定在31.3％(SB实验组)和25.8％(PB实验组)，MFI稳定在18007(SB)和9085(PB)。而SB转座子和PB转座酶交叉组、PB转座子和SB转座酶交叉组、SB和PB转座子无转座酶对照组这4个实验组细胞EGFP阳性比例和MFI值持续下降，在P5代时，细胞EGFP阳性比例降低至0.5％左右，MFI降低至3000以下，四组结果见无显著性差异。此结果证实SB和PB转座子系统能高效稳定的转染哺乳动物细胞，且两系统具有专一性。
2.使用SB和PB双转座子系统按不同质粒组合构建慢病毒生产细胞系
本实施例利用SB和PB转座子系统能高效整合基因片段在细胞基因组中，且相互不干扰的特性，将慢病毒包装所用基因rev、VSV-G、gag/pol和携带目的核酸片段的病毒基因组转录盒以及诱导表达系统激活物rtTA和/或阻遏物CymR蛋白编码序列通过不同组合，使用SB和/或PB转座子系统分一次或两次转染并进行细胞筛选的方式构建稳定生产慢病毒载体的生产细胞系,实验流程参照图3。本实施例所述组合仅为举例，本行业专业人士可以通过本实施所示方法轻易实现其它组合。本实施例使用hPGK-Luciferase-IRES-EGFP序列为目的核酸片段开发及验证本公开所述构建慢病毒稳定生产细胞系的方法，此方法在原理上不受特定目的核酸片段影响可以为任意目的核酸片段。
细胞系第一轮转染筛选实验：将293T细胞按照1.5E6个细胞每培养皿接种于60mm培养皿中培养24小时，培养基为3ml DMEM完全培养基，培养条件为37℃、5％CO 2。按照PEI方法转染细胞，操作方法如下：转染时每60mm培养皿加入500μL转染试剂，其中含有9.5μg的总质粒量，各实验组质粒加入量参照表5，总质粒量与PEI MAX(Polysciences，24765-1)的质量比为1:4，质粒与PEI MAX混合均匀，静置15分钟后加入培养皿中，转染3小时后，更换培养基为DMEM完全培养基，转染操作完成。转染24小时后，用胰酶消化细胞并将细胞全部接种于100mm培养皿(Corning，430167)中。按照表5所示筛选抗性进行细胞药物筛选，并连续在此药物压力下至少筛选3代直至细胞系稳定，其中潮霉素 (Hygromycin，生工A600230-0001)筛选浓度为200μg/ml。细胞稳定生长后移除药物并转入DMEM完全培养基中培养。
细胞系第二轮转染筛选实验：表5为细胞系第一轮转染筛选实验构建的细胞系，其中的基因改造后的人胚肾细胞系EuLV-F2于2020年4月13日以保藏编号CGMCC No.19674保藏于中国普通微生物菌种保藏管理中心(CGMCC，地址：北京市朝阳区北辰西路1号院3号)。将EuLV-F2、EuLV-F3、EuLV-F4、EuLV-F5、EuLV-F7、EuLV-F8、EuLV-F9和EuLV-F10这8条细胞系按1.5E6个细胞分别接种于8个60mm培养皿中，培养24小时。按照PEI方法转染细胞，各实验组质粒加入量参照表6。转染24小时后，用胰酶消化细胞并全部接种于100mm培养皿中。按照表6所示筛选抗性进行细胞药物筛选，并连续在此药物压力下至少筛选3代直至细胞系稳定，其中嘌呤霉素(Puromycin，阿拉丁P113126)筛选浓度为2.5μg/ml、灭瘟素(别名杀稻瘟菌素S，英文名Blasticidin-S HCl，阿拉丁B139600)筛选浓度为10μg/ml。细胞稳定生长后移除药物并转入DMEM完全培养基中培养。第二轮转染筛选实验构建了EuLV-F2-S2、EuLV-F3-S3、EuLV-F4-S4、EuLV-F5-S5、EuLV-F7-S7、EuLV-F8-S8、EuLV-F9-S9和EuLV-F10-S10共8种细胞系。其中对基因改造后的人胚肾细胞系EuLV-F2-S2细胞株进行了保藏，其于2020年4月13日以保藏编号CGMCC No.19675保藏于中国普通微生物菌种保藏管理中心(CGMCC，地址：北京市朝阳区北辰西路1号院3号)。
使用HT1080细胞Luciferase病毒滴度检测方法检测上述慢病毒稳定生产细胞系的慢病毒产毒能力。将筛选稳定后所获得的12种细胞系EuLV-NC-SB、EuLV-NC-PB、EuLV-F1、EuLV-F2-S2、EuLV-F3-S3、EuLV-F4-S4、EuLV-F5-S5、EuLV-F6、EuLV-F7-S7、EuLV-F8-S8、EuLV-F9-S9、EuLV-F10-S10按照8E+05个细胞每孔接种于6孔板(Corning 3516)中并培养在37℃、5％CO 2环境下，培养基为DMEM完全培养基。培养24小时后，更换培养基为含有诱导剂1μg/ml DOX(盐酸多西环素(DOX)，生工生物工程(上海)股份有限公司，A600889)、200μg/ml Cumate(Aladdin，I107765)和5mmol/L丁酸钠(Sigma，303410)的DMEM完全培养基诱导产毒。设置在293T细胞通过瞬时转染质粒方法所生产的慢病毒为阳性对照，具体方法如下：将293T细胞按8E+05个细胞每孔接种于6孔板中，培养24小时后加入转染试剂(总DNA量为5μg，其中摩尔比19BF081:pMD2.G(Addgene 12259):pMDLg/PRRE(Addgene 12251):pRSV-Rev(Addgene 12253)＝1:1:1:1，总DNA与PEI MAX的质量比为1:4)。转染2小时后，同上加入诱导剂(终浓度为5mmol/L丁酸钠，1μg/ml DOX，200μg/ml Cumate)。HT1080细胞Luciferase病毒滴度检测方法操作如下：在上述实验组和对照组细胞加入诱导剂24小时后，将HT1080细胞按照1E4个细胞每孔接种于96孔板(Corning 3916)中，培养基为DMEM完全培养基。诱导产毒48小时后，收集含有慢病毒的培养基并使用离心机在14000rpm离心10分钟收集病毒液上清。在加入病毒样品1小时前，将HT1080细胞的培养基更换为含有8μg/ml polybrene(Sigam，H9268)的DMEM完全培养基。之后在96孔板中每孔加入50μL的待测病毒液，阴性对照孔加入50μL DMEM完全培养基，且每个样品及对照均设置两个复 孔。培养48小时后用
Figure PCTCN2020115522-appb-000007
Luciferase Assay System(Promega，E2610)试剂盒，按照其操作说明(Promega，FB037)检测各孔相对光单位RLU(relative light unit)，检测仪器为荧光酶标仪(Perkin Elmer VictorⅤ)。
图4为按照不同SB、PB转座子系统及慢病毒包装基因组合构建的慢病毒稳定生产细胞在诱导产毒后通过HT1080细胞Luciferase病毒滴度检测方法测得的病毒转染滴度RLU值的检测结果。横坐标为不同质粒组合所构建细胞系，纵坐标为所产慢病毒在感染HT1080细胞后Luciferase的相对表达量值RLU。结果显示使用不同SB和/或PB转座子及不同慢病毒包装基因组合都能快速构建慢病毒稳定生产细胞系，各种组合所构建的稳定细胞系在诱导后都能明显检测到病毒滴度；而在转染中无转座酶对照NC-SB和NC-PB细胞在筛选后无法检测到病毒滴度，其RLU值和背景对照NC无显著区别。在构建慢病毒稳定生产细胞系时，一般会先构建含有诱导表达系统激活物和/或阻遏物以及慢病毒包装基因rev、VSV-G、gag/pol的包装细胞，然后再基于此包装细胞构建含有携带目的核酸片段的慢病毒基因组转录盒的最终慢病毒稳定生产细胞。由于EuLV-F2-S2细胞显示出最优的慢病毒载体包装能力(4.68E+05 RLU)，本公开优选先以SB转座子系统构建含有Tet-ON诱导系统反式激活物rtTA编码序列和/或Cumate诱导系统阻遏物CymR编码序列以及慢病毒包装基因rev、VSV-G、gag-pol基因的慢病毒载体包装细胞系(EuLV细胞)，再以PB转座子系统在以上慢病毒载体包装细胞系基因组中稳定插入携带目的核酸片段的慢病毒基因组转录盒的方法构建慢病毒稳定生产细胞系。
表5.细胞系构建第一轮细胞转染实验质粒信息表
Figure PCTCN2020115522-appb-000008
表6.细胞系构建第二轮细胞转染质粒信息表
Figure PCTCN2020115522-appb-000009
Figure PCTCN2020115522-appb-000010
实施例3：慢病毒基因rev、VSV-G和gag/pol的诱导表达优化
本实施例针对表达rev、VSV-G和gag/pol基因的诱导表达进行优化。主要优化条件包括三个主要方面(1)Tet-On诱导系统反式激活物rtTA的选择；(2)诱导表达系统优化包括基于Tet-On诱导系统的TRE adv、TRE 3G响应元件的单调控优化和基于Tet-On和Cumate复合诱导表达系统的TRE advCuO、TRE 3GCuO响应元件的复合调控优化；(3)以及启动子或诱导表达系统响应元件3’端和被调控核酸序列5’端之间是否连接可剪切的内含子。基于以上3个方面，本实施设计测试了8种诱导响应元件和内含子的组合以及两种Tet-On反式激活物rtTA adv和rtTA 3G来调控慢病毒包装基因gag/pol、VSV-G和rev的表达，并基于各种条件在慢病毒产毒滴度和泄露滴度这两个方面进行优化。详细质粒构建信息参照实施例1说明，具体实施步骤如下：
1.构建293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞
293T细胞培养、PEI转染以及潮霉素筛选等实验流程参照实施例2。将293T细胞按照1.5E+06个细胞每60mm培养皿接种，在37℃、5％CO 2的环境下，培养在添加10％FBS(ExCell，11H116)的DMEM(Sigam，D6429)完全培养基中。培养24小时后按照PEI方法进行转染，总质粒量为5.5μg，其中按照质粒19BF074:18BF019摩尔比10:1进行转染以获得293T-rtTA adv-CymR细胞；按照质粒19BF075:18BF019摩尔比10:1进行转染以获得293T-rtTA 3G-CymR细胞。转染后用200μg/ml潮霉素药物筛选至少三代后，细胞在药物压力下生长和原始293T细胞一致后，进行以下实验。
2.单质粒替换方法筛选优化rev、VSV-G和gag/pol的基因表达调控方式
在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内，以实施例1中构建的8种rev质粒、8种VSV-G质粒和9种gag/pol质粒分别替换表达rev的pRSV-Rev(Addgene，#12253)、表达VSV-G的pMD2.G(Addgene，#12259)和表达gag/pol的pMDLg/pRRE(Addgene，#12251)质粒，以19BF081质粒为携带目的核酸片段的病毒基因组转录盒转移载体质粒，通过PEI瞬时转染的方式分别在有无DOX和Cumate诱导剂的情况下制备病毒，并且通过HT1080细胞Luciferase病毒滴度检测方法评估产毒滴度和泄露滴度。每个实验条件仅替换一个可诱导表达的测试质粒，其它共转的质粒为非诱导表达质粒。其中8种rev测试质粒为18BF072、18BF071、19BF249、19BF248、19BF247、19BF246、18BF070和18BF069；8种VSV-G测试质粒为18BF068、18BF067、19BF245、19BF244、19BF243、19BF242、18BF066和18BF065；9种gag/pol测试质粒为18BF074、19BF131、19BF130、19BF251、19BF250、19BF129、19BF128、18BF076和19BF126；阳性对照为质粒为pMD2.G、pRSV-Rev和pMDLg/pRRE。实验流程如下：
将筛选稳定的293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞分别按1.5E+05个细胞每孔接种于24孔板中，培养体积为500μl。培养24h后，按照PEI方法进行转染，每孔加入50μl转染混合物，其中含有0.8μg的总质粒量和3.2μg的PEI。在0.8μg的总质粒量中，相应的rev、VSV-G、gag/pol和19BF081 质粒的混合摩尔比是1:1:1:1。在rev优化实验组中仅用本公开设计载体替换pRSV-Rev质粒；在VSV-G实验组中仅替换pMD2.G质粒；在gag/pol实验组中仅替换pMDLg/pRRE质粒；阳性对照组使用pMD2.G、pRSV-Rev和pMDLg/pRRE质粒转染制备慢病毒。将质粒和PEI混合均匀，静置15分钟后加入24孔板中，每个样品设置两个孔。转染3h后，更换培养基为含有5mmol/L丁酸钠的完全培养基，并在每个样品的其中一个孔中加入终浓度为1μg/ml DOX和200μg/ml Cumate诱导剂作为诱导组，另一孔加入等量培养基作为非诱导组。继续培养48h后，收集每孔病毒上清液，按照实施例2中基于HT1080细胞的Luciferase病毒滴度检测方法检测每个样品在诱导和非诱导条件下的RLU值，结果如图5所示。
图5为rev(图5A)、VSV-G(图5B)和gag/pol(图5C)各慢病毒包装基因在293T-rtTA adv-CymR或293T-rtTA 3G-CymR细胞中、被不同诱导表达系统响应元件调控以及响应元件3’端和被调控核酸序列5’端之间是否连接可剪切的内含子的条件下的诱导产毒滴度和无诱导泄露滴度的Luciferase滴度检测结果。
基于诱导产毒滴度和无诱导泄露滴度结果，rev的优选调控元件为TRE advCuO(18BF070)，TRE advCuO-BGI(19BF249)，TRE 3G(19BF246)，TRE 3GCuO(19BF247)和TRE 3GCuO-BGI(18BF072)，其中TRE 3G，TRE 3GCuO和TRE 3GCuO-BGI为优选，所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为(1)TRE 3G(19BF246)：6.80E+05 RLU,135倍和9.24E+05 RLU，322倍；(2)TRE 3GCuO(19BF247):6.34E+05 RLU，275倍和7.79E+05 RLU,279倍；(3)TRE 3GCuO-BGI(18BF072)：7.46E+05 RLU，105倍和4.96E+05 RLU，68倍。其中Tet-On单诱导系统进一步优选反式激活物为rtTA 3G，优选响应元件为TRE 3G；Tet-On和Cumate复合诱导进一步优选响应元件为TRE 3G、TRE 3GCuO和TRE 3GCuO-BGI。
基于诱导产毒滴度和无诱导泄露滴度结果，VSV-G的优选调控元件为TRE advCuO(18BF066)，TRE advCuO-BGI(19BF245)，TRE 3G-BGI(18BF067)和TRE 3GCuO-BGI(18BF068)所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为(1)TRE advCuO(18BF066)：3.34E+05 RLU，1323倍和3.35E+05 RLU,974倍；(2)TRE advCuO-BGI(19BF245)：5.96E+05 RLU，506倍和4.62E+05 RLU,886倍；(3)TRE 3G-BGI(18BF067)：4.22E+05 RLU，110倍和4.27E+05 RLU,629倍；和(4)TRE 3GCuO-BGI(18BF068)：8.09E+05 RLU，1539倍和5.76E5RLU,1260倍。其中Tet-On单诱导系统进一步优选反式激活物为rtTA 3G，优选响应元件为TRE 3G-BGI；Tet-On和Cumate复合诱导进一步优选响应元件为TRE advCuO-BGI、TRE 3G-BGI、TRE 3GCuO-BGI，再进一步优选TRE 3GCuO-BGI。
基于诱导产毒滴度和无诱导泄露滴度结果，在响应元件3’端和gag/pol编码序列5’端之间没有连接内含子(BGI)序列的条件下，无论是诱导组还是非诱导组，检测滴度都在1000 RLU以下，接近背景值；CMV启动子在下游没有内含子的情况下(19BF126)，检测滴度也下降到有内含子(18BF074)的19.4％，说明启动子和gag/pol编码序列之间连接的可剪接的内含子序列是gag/pol编码序列高表达的重要条件。转染设计含有内含子的gag/pol质粒所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为(1)TRE adv-BGI(19BF250)：8.60E+05 RLU，87倍 和6.43E+05 RLU，135倍；(2)TRE advCuO-BGI(19BF251)：9.01E+05 RLU，413倍和6.21E+05 RLU，326倍；(3)TRE 3G-BGI(19BF130)：7.53E+05 RLU，257倍和7.16E+05 RLU，333倍；和(4)TRE 3GCuO-BGI(19BF131)：8.22E+05 RLU，641倍和8.47E+0.5 RLU，692倍；(5)CMV-BGI(18BF074)：1.28E+06RUL和1.10E+06 RLU。因为gag/pol编码序列的表达同时受rev蛋白调控，所以gag/pol编码序列的调控非必须。基于以上结果，gag/pol编码序列的调控元件优选为CMV-BGI、TRE adv-BGI、TRE advCuO-BGI、TRE 3G-BGI和TRE 3GCuO-BGI。其中Tet-On单诱导优选调控元件为TRE 3G-BGI和CMV-BGI；Tet-On和Cumate复合诱导优选响应元件为CMV-BGI、TRE adv-BGI、TRE advCuO-BGI、TRE 3G-BGI和TRE 3GCuO-BGI，进一步优选CMV-BGI、TRE advCuO-BGI、TRE 3GCuO-BGI，再进一步优选CMV-BGI和TRE 3GCuO-BGI。对于以上关于gag/pol编码序列的启动子的选择的结果，本领域技术人员可以合理的预期，当使用除了CMV启动子以外的本领域中常用的其他真核启动子(例如，PGK、CAGGS、EF1a、RSV、SV40等)和除了BGI以外的本领域中常用的其他内含子的组合来调控gag/pol的转录时，可以取得与CMV-BGI的设计方案相似的结果。
3.基于以上优选调控元件综合优化rev、VSV-G和gag/pol表达质粒组合
根据以上单质粒替换方法筛选优化rev、VSV-G和gag/pol基因表达的实验结果，选取(1)rev：18BF072、19BF247和19BF246；(2)VSV-G：18BF068和18BF067；(3)gag/pol：18BF074、19BF131和19BF130质粒按照表7所示组合在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内，按照上述24孔板瞬时转染制备慢病毒方法，分别在有无DOX和Cumate诱导剂的情况下制备病毒，并且通过HT1080细胞Luciferase病毒滴度检测方法评估产毒滴度和泄露滴度。细胞接种、质粒加入量及各质粒摩尔比例、PEI转染、转染换液、诱导产毒和HT1080细胞Luciferase滴度检测等实验条件同本实施例单质粒替换筛选实验一致，结果如图6所示。
如图6结果所示：基于诱导产毒滴度和无诱导泄露滴度结果，Tet-On单诱导优选质粒组合为rev：19BF246、VSV-G：18BF067和gag/pol：18BF074或19BF130。在使用18BF074质粒时，所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为6.83E+05 RLU，45倍和7.28E+05 RLU，167倍；在使用19BF130质粒时，所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为5.20E+05 RLU，24倍和6.94E+05 RLU，176倍。实验结果所示，18BF074和19BF130质粒在诱导RLU平均值和诱导/泄露产毒滴度比值上无显著差距，而rtTA 3G相比rtTA adv反式激活物可以显著降低非诱导泄露。
如图6结果所示，Tet-On和Cumate复合诱导优选质粒组合最优三个诱导后产毒滴度RLU平均值来自样品编号19BF246+18BF068+19BF131、19BF246+18BF068+18BF074和19BF247+18BF068+18BF074质粒组合。在使用19BF246+18BF068+19BF131质粒组合时，所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为1.28E+06 RLU，1935倍和1.46E+06RLU，12032倍；在使用19BF246+18BF068+18BF074质粒组合时，所对应的诱导RLU平均值和诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为8.75E+05 RLU，262倍和1.07E+06 RLU，975倍；在使用19BF247+18BF068+18BF074质粒组合时，所对应的诱导RLU平均值和 诱导/泄露产毒滴度比值在293T-rtTA adv-CymR和293T-rtTA 3G-CymR细胞内分别为1.05E+06 RLU，563倍和1.09E+06 RLU，839倍。基于以上三组数据和图2数据所示，rev优选质粒为19BF246或19BF247，进一步优选为19BF246；VSV-G优选质粒为18BF068；gag/pol优选质粒为19BF131和18BF074，进一步优选为19BF131；反式激活物优选为rtTA 3G。
表7.rev、VSV-G和gag/pol表达质粒转染组合
Figure PCTCN2020115522-appb-000011
实施例4：优化rtTA-CymR、rev、VSV-G和gag/pol片段在细胞基因组中稳定整合的比例
慢病毒的高效生产同时需要诱导后的高病毒基因表达量和恰当的各病毒基因的的相对表达比例。各基因的表达量主要受启动子或诱导响应元件的效率和在细胞基因组整合的拷贝数影响。转座子系统在细胞基因组中稳定整合目的核酸片段的效率受基因片段长度影响，越长的目的核酸片段整合效率越低，因此可以根据目的核酸片段长度调整构建稳定细胞系时各转座子质粒在瞬时转染时比例来优化各片段的整合比例。本实施例以实施例3优化的其中一组优选质粒组合19BF075、19BF246、18BF068和19BF131质粒为例，先通过调整rev、VSV-G和gag/pol单个包装基因质粒在细胞系构建瞬时转染的摩尔比例来确认转染质粒加入量和在宿主细胞基因组整合的拷贝数的关系以及对应构建细胞的产毒能力；再通过组合优化单个或多个关键基因在细胞系构建瞬时转染的摩尔比例确认最佳基因组整合拷贝数和摩尔比例。基 于本实施例所选质粒确认的最优基因组整合比例可以根据实施例3所示各启动子/响应元件在诱导后的产毒效率或以其它本公开未囊括的启动子/响应元件的对比表达效率推导出其它质粒设计和组合的优化插入拷贝数和摩尔比例，因此也在本实施例佐证的权利要求保护范围。
1.调整单基因插入比例对包装细胞系的产毒影响
细胞培养、PEI转染以及潮霉素药物筛选等实验流程参照实施例2。将293T细胞按照1.5E+06个细胞每60mm培养皿接种，在37℃、5％CO2的环境下，培养在添加10％FBS(ExCell，11H116)的DMEM(Sigma，D6429)完全培养基中。培养24小时后以PEI方法并且按照表8所示各质粒含量进行转染(PEI和DNA质量比4：1)。转染后用200μg/ml潮霉素药物至少筛选细胞三代至细胞系稳定生长，构建EuLV-R1至R17共17种慢病毒包装细胞系。在各细胞系稳定后，参照实施例3中，24孔板制备病毒方法，将EuLV-R1至R17细胞系以1.5E+05个细胞每孔接种于24孔板中，以19BF081质粒为转移载体质粒，通过PEI瞬时转染的方式转染上述EuLV-R1至R17细胞(19BF081质粒：0.28μg、空质粒18BF003：0.52μg，总质粒0.80μg。PEI和DNA质量比4：1)。转染6小时后更换培养基并加入诱导剂(终浓度为1μg/ml DOX，200μg/ml Cumate和5mmol/L丁酸钠)诱导产毒。48小时后收集培养基上清液通过HT1080细胞Luciferase病毒滴度检测方法检测EuLV-R1至R17各细胞产毒滴度。
将EuLV-R1至R17按1.5E+05个细胞每孔接种于24孔板中，培养48小时后收集细胞抽提基因组DNA并用qPCR方法绝对定量EuLV-R1至R5细胞系基因组中整合rtTA和CymR基因的拷贝数；EuLV-R1和EuLV-R6至R9细胞系基因组中整合rev基因的拷贝数；EuLV-R1和EuLV-R10至R13细胞系基因组中整合VSV-G基因的拷贝数；EuLV-R1和EuLV-R14至R17细胞系基因组中整合gag/pol基因的拷贝数，具体方法如下。收集上述细胞系各1.0E+06个细胞，并依照基因组DNA纯化试剂盒(TIANGEN，DP304-03)说明提取基因组gDNA，将纯化后gDNA用试剂盒洗脱缓冲液调整到50ng/μl。将质粒19BF074、18BF072、18BF068和18BF074分别做为HygroR(检测rtTA和CymR拷贝数)、rev、VSV-G和gag/pol基因的标准样品，分别用去离子水稀释到47.9ng/μl、23.8ng/μl、30.0ng/μl和47.6ng/μl，此浓度下的各质粒对应5.0E+09拷贝数每微升样品。将4个标准样品进一步稀释到8.0E+06拷贝数每微升，并以此样品按两倍梯度稀释直到1.56E+04拷贝数每微升，将4个标准品的此10个稀释样品做为各个待检基因的qPCR标准曲线。将上述细胞gDNA样品和各基因qPCR标准曲线样品各2μl加入qCPR反应试剂，包含10μl NovoStart Probe qPCR SuperMix和ROX I染料0.4μl(Novoprotein Scientific Inc.E091-01A)，浓度为10μM正向、反向引物和Taqman探针各0.4μl(合成自通用生物系统(安徽)有限公司，具体引物探针序列信息见表10，其中HygroR的正向、反向引物和探针分别为：SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53；rev的正向、反向引物和探针分别为：SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56；VSV-G的正向、反向引物和探针分别为：SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59；gag/pol的正向、反向引物和探针分别为：SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50)，并补水至20μl。在ABI7900实时PCR检测仪上，选择AQ程序并依照95℃ 5分钟起始，95℃ 30秒-60℃ 30秒-72℃ 30秒40个循环然后60℃ 30秒的条件进行PCR反应并基于标准曲线和样品CT值计算各样品中待测基因的拷贝数，所得数据再按照每细胞6pg基因组DNA计算每细胞基因拷贝数。
实验结果如图7所示，横坐标表示构建细胞时转染4质粒的摩尔比例；柱状图对应左侧纵坐标显示各细胞诱导产毒滴度RLU值；线图对应右侧纵坐标显示各细胞基因组中整合HygroR(检测rtTA和CymR拷贝数)、rev、VSV-G或gag/pol片段的拷贝数结果。结果显示(1)增高rtTA 3G-CymR(HygroR探针实验结果)和rev片段在细胞基因组的整合拷贝数对EuLV包装细胞系的病毒制备能力没有明显影响(图7A和7B)。(2)将VSV-G的基因组整合拷贝数从1拷贝每细胞提高至2拷贝每细胞时，产毒滴度从6.5E+05 RLU显著上升至1.1E+06 RLU。但随着VSV-G基因组整合拷贝数达到4拷贝每细胞或以上时，产毒滴度逐渐下降，并且在基因组整合拷贝数达到6拷贝每细胞时降到3.9E+05 RLU(图7C)。说明诱导表达后VSV-G的细胞毒性可能随着基因组整合拷贝数的提高而提高，从而影响EuLV包装细胞系的产毒能力。(3)gag/pol片段的整合拷贝数正向影响EuLV包装细胞系的产毒能力，产毒滴度随着gag/pol基因组插入拷贝数提高而提高，在达到4.7拷贝每细胞以上时达到最高的产毒滴度1.6E+06 RLU，继续提高插入拷贝数至6以上无法进一步提高产毒能力(图7D)。
2.进一步调整VSV-G和gag/pol的基因组整合拷贝数和相对比例优化EuLV包装细胞系产毒能力
基于以上结果，VSV-G和gag/pol片段在细胞基因组的整合拷贝数和摩尔比例是优化EuLV包装细胞系产毒能力的关键，需要进一步优化VSV-G和gag/pol片段的转染总量和摩尔比例。同上述使用SB转座子系统构建EuLV包装细胞系方法一致，基于表9所示各质粒含量进一步构建EuLV-VG1至VG5包装细胞系。在各细胞系经潮霉素筛选至稳定生长后，同上述24孔板慢病毒制备方法一致，以19BF081质粒为目的基因质粒制备病毒并通过HT1080细胞检测luciferase病毒滴度；同时如上述qPCR定量方法，检测各细胞系基因组DNA中每细胞VSV-G和gag/pol片段的整合拷贝数，实验结果如图8所示。
实验结果如图8所示，横坐标表示构建细胞时转染4质粒的摩尔比例；柱状图对应左侧纵坐标显示各细胞诱导产毒滴度RLU值；线图对应右侧纵坐标显示gag/pol和VSV-G片段在细胞基因组中整合拷贝数的比值，结果如下。在EuLV-VG1至VG5细胞中，当gag/pol片段在细胞基因组中整合拷贝数达到5.2个拷贝每细胞，gag/pol和VSV-G片段在细胞基因组中整合拷贝数比值在2.73时(EuLV-VG2)，达到最大产毒滴度2.8E+06 RLU。继续提高gag/pol和VSV-G片段整合拷贝数比值将降低包装细胞的产毒能力。基于此结果，gag/pol片段在细胞基因组中整合拷贝数2至8个拷贝数每细胞，gag/pol和VSV-G片段在细胞基因组中整合拷贝数比例在1:1至4:1之间，rtTA 3G-CymR和rev片段至少在细胞基因组中整合一个拷贝数的情况下可以得到高产毒滴度的包装细胞系；进一步优选，gag/pol片段在细胞基因组中整合拷贝数在4至6个拷贝数每细胞，gag/pol和VSV-G片段整合比例在2:1至3:1之间，rtTA 3G-CymR和rev片段至少在细胞基因组中整合一个拷贝数的情况下可以得到高产毒滴度的包装细胞系。
表8.构建EuLV-R1至R17包装细胞系瞬时转染各质粒含量
Figure PCTCN2020115522-appb-000012
Figure PCTCN2020115522-appb-000013
表9.构建VG1至VG9细胞系瞬时转染各质粒含量
Figure PCTCN2020115522-appb-000014
表10.引物和Taqman探针序列的信息表
Figure PCTCN2020115522-appb-000015
实施例5：EuLV293T3rd慢病毒包装细胞系构建及高通量单克隆筛选
EuLV293T3rd慢病毒包装细胞系构建流程如图9所示，包括质粒转染及细胞筛选，单克隆筛选和悬浮适应3个步骤。实验细节及结果如下详细描述。
1.通过质粒转染及细胞系筛选构建初始EuLV293T3rd慢病毒包装细胞系
具体实验方法如下。细胞培养、PEI转染以及潮霉素筛选等实验流程参照实施例2、3和4。将293T(ATCC，CRL3216)细胞按1.5E+06个细胞每培养皿接种于60mm培养皿中，培养基为3ml添加10％FBS(ExCell，11H116)的DMEM(Sigma，D6429)完全培养基，在37℃5％CO 2条件下培养24小时。以PEI转染法进行质粒转染，总质粒量为5μg，PEI和总质粒质量比为4:1,其中按照质粒19BF257:19BF246:18BF067:19BF130:18BF019摩尔比例为3:3:2:12:2进行转染构建EuLV293T3rd-SB16细胞；按照质粒19BF075:19BF246:18BF068:19BF131:18BF019摩尔比例为3:3:2:12:2进行转染构建 EuLV293T3rd-SB28。转染24小时后，将细胞全部接种至100mm培养皿中继续培养，并加入200μg/ml潮霉素(生工A600230-0001)进行筛选。维持此筛选压力至少传代培养3代至细胞系稳定，判断依据为：(1)细胞生长速度与原始293T细胞一致；(2)SB转座酶表达质粒18BF019在构建细胞中基本消失，表现为ECFP阳性细胞比例跌至1％以下；(3)细胞活率在95％以上。基于以上标准筛选得到Tet-On单诱导表达系统调控的EuLV293T3rd-SB16(简称SB16细胞)和Tet-On和Cumate复合诱导表达系统调控的EuLV293T3rd-SB28(简称SB28细胞)初始细胞系。
2.筛选EuLV293T3rd-SB16和EuLV293T3rd-SB28高产单克隆包装细胞株
具体实验方法如下。SB16和SB28高产单克隆细胞株筛选流程如图9所示，首先将SB16和SB28细胞以极限稀释法按照1个细胞每孔接种至共10块384孔板进行首轮筛选，筛选依据为：(1)有且只有一个单克隆细胞；(2)细胞形态正常；(3)细胞生长速度与原始293T细胞相同。然后以HT1080细胞Luciferase病毒滴度检测法分别在细胞扩增至96孔板、24孔板、和6孔板培养体系时检测各单克隆细胞诱导产毒滴度。
图10为各筛选阶段单克隆细胞产毒滴度与标准品比值的分布情况。按照HT1080细胞Luciferase病毒检测方法检测单克隆细胞产毒滴度RLU值并按照500为间隔在0至100000之间统计每个区间范围内单克隆数，横坐标为每个区间的上限值与标准品检测RLU值的比值(以2为底的对数表示)，纵坐标为各统计范围内单克隆细胞数。图10A为SB16细胞系筛选结果，分别在96孔板、24孔板和6孔板筛选步骤中选出94、30和9株单克隆细胞，图10B为SB28细胞系筛选结果，分别在96孔板、24孔板和6孔板筛选步骤中选出87、30和9株单克隆细胞。细胞筛选具体操作方法如下所述。
384孔板单克隆筛选，具体实验步骤如下。以DMEM完全培养基在37℃，5％CO 2条件下分别培养上述构建的SB16与SB28初始细胞细胞至单层70％汇合度。使用有限稀释法，按照每孔一个细胞的密度将细胞接种于384孔板中，SB16和SB28两种细胞各接种5块384孔板。接种2小时待细胞贴壁后，利用倒置显微镜(Olympus IX71)观察并标记有且只有一个细胞的样品孔，舍弃其他样品孔。接种后第5天和第9天更换新鲜的DMEM完全培养基，第10天将汇合度大于50％的单克隆细胞按每孔2E+04个细胞接种至96孔板中，共获得SB16单克隆细胞271株和SB28细胞单克隆313株。
96孔板单克隆筛选，具体实验步骤如下。将384孔板单克隆筛选步骤所得细胞培养24小时后，按照每孔5E+03和2.5E+04个细胞接种于两块新的96孔板中，分别用于传代培养和慢病毒诱导产毒检测。在单克隆细胞诱导产毒步骤中，设置通过瞬时转染293T细胞方法制备慢病毒阳性对照，方法如下：将293T细胞按2.5E+04个细胞每孔接种于96孔板中，培养24小时后通过PEI方法瞬时转染质粒制备病毒(总DNA量为0.3μg，其中质粒19BF081:pMD2.G:pMDLg/PRRE:pRSV-Rev的摩尔比例为1:1:1:1，总DNA与PEI MAX的质量比为1:4)。转染2小时后，加入诱导剂(终浓度为2mmol/L丁酸钠，1μg/ml DOX，200μg/ml Cumate)，并继续培养48小时后，通过4500rpm离心15分钟方法收集上清病毒。96孔板单克隆细胞诱导产毒方法如下：将上述按2.5E+04个细胞每孔接种于96孔板的待测单克隆细胞继续培养24小时后，加入转染试剂(总DNA量为0.3μg，其中19BF081加入量和上述阳性对照一致，剩余质粒量用18BF003补至0.3μg，总DNA与PEI MAX的质量比为1:4)。之后诱导产毒及回收病毒上清液的方法和上述阳性对照制备方法一致。按照实施例2所述HT1080细胞Luciferase病毒滴度检测方法检测阳性对照和各单克隆细胞诱导产毒样品的RLU值。之后根据Luciferase病毒滴度结果，从高到低依次挑选出RLU值高于阳性对照的单克隆细胞，获得SB16单克隆细胞87株和SB28细胞单克隆94株。
24孔板和6孔板单克隆筛选，具体实验步骤如下。24孔板与6孔板高产单克隆细胞筛选流程同上述96孔板单克隆筛选基本一致。其中传代时，24孔板接种密度为每孔2E+04个细胞，6孔板为2E+05个细胞。在单克隆细胞诱导产毒步骤中，24孔板接种密度为每孔1E+05个细胞，6孔板接种密度为8E+05个细胞。用PEI方法转染质粒时，24孔板每孔总DNA量为1.2μg，6孔板为5μg，其它转染条件如各质粒比例及DNA与PEI的比例与上述实验方法一致。根据HT1080细胞Luciferase病毒滴度检测方法结果，在24孔板单克隆筛选阶段，进一步从87株SB16和94株SB28单克隆细胞中各筛选30株高产包装细胞株。在6孔板单克隆筛选阶段，又从各自30株细胞中进一步筛选出SB16和SB28单克隆高产包装细胞各9株，最终筛选单克隆细胞编号如表11所示。
表11.高产包装细胞克隆编号列表
EuLV293T 3rd-SB16高产克隆编号 EuLV293T 3rd-SB28高产克隆编号
EuLV293T 3rd-SB16-3A5 EuLV293T 3rd-SB28-1A3
EuLV293T 3rd-SB16-3C1 EuLV293T 3rd-SB28-1C2
EuLV293T 3rd-SB16-3C3 EuLV293T 3rd-SB28-1C3
EuLV293T 3rd-SB16-3C4 EuLV293T 3rd-SB28-1C4
EuLV293T 3rd-SB16-3D3 EuLV293T 3rd-SB28-1C6
EuLV293T 3rd-SB16-4A1 EuLV293T 3rd-SB28-2B5
EuLV293T 3rd-SB16-4A5 EuLV293T 3rd-SB28-2C3
EuLV293T 3rd-SB16-4B1 EuLV293T 3rd-SB28-2C6
EuLV293T 3rd-SB16-4C4 EuLV293T 3rd-SB28-2D1
3.高产EuLV293T3rd单克隆包装细胞悬浮适应及病毒包装能力检测
具体实验方法如下。将表11中得到的18株单克隆细胞按5E+05细胞/ml，每瓶20ml接种至125ml摇瓶(Corning 431143)中进行悬浮适应，培养基为Freestyle293(
Figure PCTCN2020115522-appb-000016
FreeStyle TM 293Expression Medium 12338018)，培养条件为37℃，5％CO 2，1.9cm摇距摇床，转速140rpm。细胞生长至2.5E+06细胞/ml时进行传代，连续培养至无明显细胞结团并且倍增时间小于24小时后，得到上述18株细胞的悬浮培养细胞系。悬浮培养的SB16和SB28细胞系的病毒制备方法如下。将上述细胞悬浮培养至2.5E+06细胞/ml密度后，以1000rpm离心3分钟收获细胞，并用新鲜的Freestyle293培养基重悬并调整细胞密度至4E+06细胞/ml。将细胞悬液按每孔0.5ml转移至96深孔板中，加入以DMEM培养基配制的50μL PEI转染试剂，其中包含7.5μg PEI MAX和2.5μg总DNA(其中含有1μg 18BF081和1.5μg 18BF003)。混匀后在培养摇床上继续培养，培养条件为37℃，5％CO2，1.0cm摇距摇床，转速1000rpm。两小时后，加入诱导剂(终浓度为2mmol/L丁酸钠，1μg/ml DOX，200μg/ml Cumate)，继续培养48小时后，通过1500rpm离心15分钟并收集上清病毒。之后依照实施例2所述HT1080细胞Luciferase病毒滴度检测方法检测病毒滴度。检测结果如图11所示，横坐标为单克隆编号，纵坐标为滴度检测结果，以RLU表示。结果显示所有单克隆细胞都能适应无血清悬浮培养条件，不同单克隆细胞在悬浮培养条件下瞬时转染19BF081质粒的产毒滴度在9.96E+04至4.68E+06 RLU之间，平均滴度为8.56E+05 RLU，中位值为5.7E+05 RLU。
实施例6：EuLV293T3rd最优产毒包装细胞株优化程度检测
将实施例5中筛选出的最优产毒单克隆包装细胞株，在通过瞬时转染转移载体质粒包装慢病毒的同时，共转其它由组成型活性启动子调控的rev、gag/pol、VSV-G慢病毒包装基因表达质粒，检测继续增加病毒包装基因的表达能否进一步增加包装细胞的产毒滴度，从而判断细胞株中各包装基因片段是否已达到饱和以及各病毒包装基因片段相对比例是否达到最优化。从实施例5中选取6株高产贴壁细胞株和5株高产悬浮细胞株进行上述实验。细胞株名称编号如表12所示。
表12.EuLV293T3rd优选高产贴壁培养细胞株和悬浮培养细胞株编号
序号 贴壁培养高产细胞株编号 悬浮培养高产细胞株编号
1 EuLV293T3rd-SB16-3A5 Sus-EuLV293T3rd-SB16-3C3
2 EuLV293T3rd-SB16-3C3 Sus-EuLV293T3rd-SB16-3D3
3 EuLV293T3rd-SB16-4A5 Sus-EuLV293T3rd-SB16-4A5
4 EuLV293T3rd-SB28-1A3 Sus-EuLV293T3rd-SB28-1A3
5 EuLV293T3rd-SB28-1C2 Sus-EuLV293T3rd-SB28-1C2
6 EuLV293T3rd-SB28-2C6  
贴壁培养高产细胞株慢病毒包装基因补充实验：将表12所示贴壁高产细胞株培养在37℃、5％CO 2环境下，培养基为DMEM完全培养基。之后将每种细胞分别按照8E+05个细胞每孔接种于6孔板的不同孔中，依照实施例2中磷酸钙转染方法进行转染实验。转染时每孔加入200μL转染试剂，含有0.12mol/L的氯化钙，1xHEPES缓冲液，和6.8μg的总质粒量。各实验组质粒加入组合及含量参照表13，其中18BF084质粒表达rtTA adv反式激活物；19BF081为转移质粒转录携带hPGK-Luciferase-IRES-EGFP目的核酸片段的慢病毒基因组；pMD2.G(Addgene，12259)表达VSV-G蛋白；pMDLg/pRRE表达gag和pol蛋白；pRSV-Rev表达rev蛋白。转染6小时后，更换培养基为含有5mM丁酸钠的DMEM完全培养基并按照表13中需要加入诱导剂的样品中加入1μg/ml DOX和200μg/ml Cumate诱导剂。继续培养48小时后，按照实施例2所述HT1080细胞Luciferase病毒滴度检测方法检测各贴壁细胞株慢病毒产毒能力，结果如图12所示。横坐标为各实验组质粒转染组合，纵坐标为对应实验组感染HT1080细胞表达Luciferase的RLU值。图中虚线标注为该贴壁细胞株在此条件下仅瞬转19BF081转移质粒诱导产毒的滴度结果RLU值，图中实线为293T的阳性对照结果。结果表明：6株包装细胞在未补充慢病毒包装基因时，转染滴度在9.9E+05 RLU至5.8E+06 RLU之间。在补充rev、VSV-G、gag/pol等慢病毒包装基因后，多数高产细胞株(如EuLV293T3rd-SB28-1A3和EuLV293T3rd-SB28-1C2)的产毒滴度没有进一步显著增长。而产毒能力提升较大的细胞都是初始产毒滴度较低的细胞。多数细胞株在进一步补充rtTA、rev、VSV-G、gag/pol单一基因或多个基因后产毒滴度都有不同程度下降，仅有部分细胞株受益于进一步补充gag/pol基因，说明多数筛选所得细胞株的包装基因比例已达到充分优化，再进一步补充病毒包装基因反而会降低细胞株产毒滴度，另外也再次证明gag/pol的插入拷贝数以及表达量对细胞株产毒滴度的重要性。
悬浮培养高产细胞株包装基因补充实验：将表12所示悬浮细胞株培养在37℃、8％CO 2，摇床摇距为1.9cm，转数为140rpm的环境中，培养基为FreeStyle(
Figure PCTCN2020115522-appb-000017
FreeStyle TM 293 Expression Medium，12338018)。在转染前2小时，将各悬浮细胞株按照4E+06个细胞每孔接种于96孔深孔板中，培养体积为1ml。按照实施例5中PEI方法转染细胞，转染时每孔加入100μL转染样品，含有2.5μg的总质粒量，7.5μg的PEI。各实验组质粒加入量参照表14。转染后将深孔板培养在摇距为1mm，转数为1000rpm/min的环境中。转染3小时后，加入终浓度为2mM的丁酸钠，并按表14中需要加入诱导剂的样品中加入1μg/ml DOX和200μg/ml Cumate诱导剂。继续培养48小时后，按照实施例2所述HT1080细胞Luciferase慢病毒滴度检测方法检测各悬浮细胞的产毒能力。结果如图13所示。横坐标为各实验组质粒转染组合，纵坐标为对应实验组感染HT1080细胞表达Luciferase的RLU值。图中虚线标注为该悬浮细胞株在此条件下仅瞬转19BF081转移质粒诱导产毒的滴度结果RLU值，图中实线为293T的阳性对照结果。结果 表明：5株悬浮细胞在未补充慢病毒包装基因时，RLU结果在8.9E+05 RLU至4.0E+06 RLU之间。在补充rev、VSV-G、gag/pol等慢病毒包装基因后，多数高产细胞的产毒滴度没有进一步显著增长。而产毒能力提升较大的细胞都是初始产毒滴度较低的细胞。多数细胞株在进一步补充rtTA、rev、VSV-G、gag/pol单一基因或多个基因后产毒滴度都有不同程度下降，仅有部分细胞受益于进一步补充gag/pol基因，说明多数筛选所得细胞株的包装基因比例已达到充分优化，再进一步补充病毒包装基因反而会降低细胞株产毒滴度。
表13.贴壁培养高产细胞株慢病毒包装基因补充实验中各实验组质粒名称及用量
Figure PCTCN2020115522-appb-000018
表14.悬浮培养高产细胞株慢病毒包装基因补充实验中各实验组质粒名称及用量
Figure PCTCN2020115522-appb-000019
实施例7：基于高产包装细胞株EuLV293T3rd-SB16-3D3和EuLV293T3rd-SB28-1C2构建多种慢病毒生产细胞系
本实施例将含有3种不同目的核酸片段的转移载体质粒分别通过PB转座子系统稳定整合在EuLV293T3rd-SB16-3D3(以下简称3D3)和EuLV293T3rd-SB28-1C2(以下简称1C2)包装细胞的基因组中，并分别筛选得到稳定生产3种不同慢病毒的6种慢病毒生产细胞系。上述3种携带目的核酸片段 的病毒基因组转录盒的转移载体质粒分别为：(1)19BF081(hPGK-Luciferase-IRES-EGFP，慢病毒基因组长度5.7kbp)；(2)19BF218(缺失B蛋白结构域的凝血因子8序列，CMV-BDDF8cHA-IRES-EGFP，慢病毒基因组长度8.5kbp)；(3)19BF217(凝血因子8全长序列，CMV-F8cHA-IRES-EGFP，慢病毒基因组长度11.2kbp)。为了方便检测以上生产细胞系的慢病毒产毒滴度，上述目的核酸片段都连接了IRES-EGFP序列，详细质粒构建方法参照实施例1。之后这些稳定慢病毒生产细胞系的产毒能力在贴壁培养条件下和相同转移质粒通过瞬时转染293T生产慢病毒方法做对比。之后选取基于3D3和1C2构建的携带19BF081转移载体的两种细胞在无血清培养基条件下进行悬浮培养适应，并在此条件下检测诱导后产毒滴度和无诱导条件下泄露产毒滴度。本公开所述构建稳定慢病毒生产细胞系的方法可用于构建任何目的核酸片段的慢病毒稳定生产细胞系。
基于3D3和1C2细胞构建稳定生产3种不同慢病毒的生产细胞系，具体实验方法如下。将3D3和1C2细胞按1.5E+06细胞每孔接种于6孔板中，培养基为3ml DMEM完全培养基，在37℃5％CO 2条件下培养24小时后，按照PEI方法进行转染，总质粒量为5.5μg，其中按照转移质粒和PB转座酶质粒(18BF031)摩尔比10:1进行转染，PEI总量为22μg。其中转移质粒分别为19BF081，19BF218和19BF217。转染后24小时加入2.5μg/ml嘌呤霉素(Puromycin，阿拉丁P113126)筛选至少3代至细胞稳定，共得到6株稳定慢病毒生产细胞系，分别为3D3-19BF081、3D3-19BF218、3D3-19BF217、1C2-19BF081、1C2-19BF218和1C2-19BF217。
稳定慢病毒生产细胞系产毒方法和瞬时转染产毒方法的产毒能力对比，具体实验方法如下。将上述筛选得到的6种细胞按8E+05细胞每孔接种于6孔板中，培养基为DMEM完全培养基，在37℃5％CO 2条件下培养24小时后更换新的DMEM完全培养基并加入诱导剂(终浓度为2mmol/L丁酸钠，1μg/ml DOX和200μg/ml Cumate)。继续培养48小时后用4500rpm离心15分钟方法收集病毒上清液。通过瞬时转染293T细胞制备病毒方法和实施例五所述阳性对照慢病毒制备方法一致，其中转移载体质粒:pMD2.G:pMDLg/PRRE:pRSV-Rev的摩尔比例为1:1:1:1，转移载体质粒分别为19BF081，19BF218和19BF217。
基于HT1080细胞EGFP阳性率方法检测以上病毒样品转染滴度，具体实验方法如下。将HT1080细胞(ATCC CCL-121)，按照5E+04个细胞每孔接种于24孔板(Corning 3524)中，培养条件与293T相同。24小时后取至少3孔用胰蛋白酶消化并计数，其他孔更换为500μL含有8μg/mL polybrene(Sigma H9268)的DMEM完全培养基。将病毒液用DMEM完全培养基稀释至多个检测浓度，然后在培养HT1080细胞的24孔板中加入100μL稀释病毒液，每种病毒样品检测多个稀释浓度，阴性对照加入100μL DMEM完全培养基，混匀后培养48小时。之后使用流式细胞仪(艾森NovoCyte Flow Cytometer)检测各孔EGFP阳性率。选择EGFP阳性率在15％～30％的稀释度按照以下公式计算慢病毒滴度。慢病毒滴度(TU/ml)＝稀释倍数×(24孔板细胞计数结果×阳性率)/0.1ml。实验结果如图14所示。结果显示稳定慢病毒生产细胞 系产毒方法的产毒滴度显著高于瞬时转染293T细胞方法的产毒滴度。基于两种不同诱导表达策略构建的3D3和1C2细胞所构建的6种最终生产细胞的产毒滴度都显著高于对应的瞬时转染293T阳性对照。携带19BF081(5.7kb)、19BF217(11.2kb)和19BF218(8.5kb)转移载体的三种3D3慢病毒稳定生产细胞系的产毒滴度分别为1.11E6TU(EGFP)/ml、2.33E5TU(EGFP)/ml和1.05E6TU(EGFP)/ml，分别是瞬时转染阳性对照的6.14、8.27和5.01倍；携带以上3种转移载体的三种1C2慢病毒稳定生产细胞系的产毒滴度分别为1.59E6TU(EGFP)/ml、3.55E5TU(EGFP)/ml和1.81E6TU(EGFP)/ml，分别是瞬时转染阳性对照的8.81、12.6和8.62倍。此外稳定慢病毒生产细胞系在表达长目的核酸片段时产毒滴度对比瞬时方法对照能高1个数量级以上，有更大的行业应用范围，比如在使用基因治疗方法治疗血友病时构建表达全长凝血因子8的慢病毒载体(以19BF217为例)。
在悬浮培养条件下检测3D3-19BF081和1C2-19BF081细胞的产毒滴度和泄露病毒滴度，具体实验方法如下。按照实施例5中所述细胞悬浮适应方法将3D3-19BF081和1C2-19BF081细胞适应悬浮培养。当悬浮细胞生长至2.5E+06细胞/ml时，用1000rp/min离心3分钟方法收获细胞，并用新鲜的Freestyle293培养基将细胞密度调整至4E+06细胞/ml。将细胞悬液按照每管5ml转移至50ml离心管中，设置加入诱导剂(终浓度为2mmol/L丁酸钠，1μg/ml DOX，200μg/ml Cumate)的诱导组和加入DMEM培养基的无诱导组，之后置于细胞培养摇床上继续培养48小时后，用4500rpm离心15分钟方法收集上清病毒。阳性对照为瞬时转染在同样悬浮培养条件培养的293T细胞的方法制备的病毒，具体方法如下。以PEI转染法进行质粒转染，总质粒量为12.5μg，PEI和总质粒质量比为4:1,其中质粒19BF081:pMD2.G:pMDLg/PRRE 1:pRSV-Rev的摩尔比例为1:1:1:1。转染6小时后，加入终浓度2mmol/L丁酸钠，并继续培养直至离心回收病毒样品。之后按照实施例2所述HT1080细胞Luciferase病毒滴度检测方法检测各样品RLU值，结果如图15所示。结果显示3D3-19BF081和1C2-19BF081细胞都能较好的适应悬浮培养条件，在诱导后分别到达7.08E6 RLU和1.08E7 RLU的病毒滴度，是瞬时转染悬浮293T细胞方法制备病毒的3.72倍和5.8倍。在无诱导条件下，两种细胞的泄露产毒滴度的RLU荧光值和不含有19BF081载体的原始细胞和293T细胞的背景RLU值基本一致，基于本实验结果测得3D3-19BF081和1C2-19BF081细胞的诱导/泄露产毒滴度比例分别为5853和41646倍。

Claims (67)

  1. 一种用于制备用于生产携带目的核酸片段的逆转录病毒载体的生产细胞的方法，所述方法包括：
    利用睡美人(Sleeping Beauty，SB)转座子系统将逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用PiggyBac(PB)转座子系统将所述逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用SB转座子系统将所述逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中。
  2. 一种用于制备用于生产携带目的核酸片段的慢病毒载体的生产细胞的方法，所述方法包括：
    利用SB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用PB转座子系统将所述慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的一种或多种但非全部整合在宿主细胞的基因组中，然后利用SB转座子系统将所述慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列中的剩余的一种或多种进一步整合在所述宿主细胞的基因组中。
  3. 根据权利要求2所述的方法，其中利用SB转座子系统将慢病毒的gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用PB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将慢病毒的gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用SB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中。
  4. 根据权利要求2所述的方法，其中所述慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列位于两个以上的构建体上。
  5. 根据权利要求4所述的方法，其中gag、pol基因的序列位于一个构建体上，rev基因的序列位于另一个构建体上，病毒包膜蛋白的编码序列位于第三个构建体上，并且携带目的核酸片段的病毒基因组转录盒序列位于第四个构建体上。
  6. 根据权利要求2所述的方法，其中所述慢病毒的gag、pol和rev基因是HIV-1病毒的gag、pol和rev基因。
  7. 根据权利要求2所述的方法，其中所述病毒包膜蛋白选自猫白血病病毒(RD114)包膜蛋白、两性逆转录病毒包膜蛋白、嗜性逆转录病毒包膜蛋白、狒狒猿白血病病毒包膜蛋白、尼帕病毒包膜蛋白、莫科拉病毒包膜蛋白、淋巴细胞脉络膜脑膜炎病毒包膜蛋白、基孔肯雅病毒包膜蛋白、罗斯河病毒包膜蛋白、塞姆利基森林病毒包膜蛋白、信德比斯病毒包膜蛋白、委内瑞拉马脑炎病毒包膜蛋白、西部马脑炎病毒包膜蛋白、流感病毒包膜蛋白、禽瘟病毒包膜蛋白、昌迪普拉病毒和皮里病毒包膜蛋白、猿免疫缺陷病毒包膜蛋白、猫免疫缺陷病毒包膜蛋白、马传染性贫血病毒包膜蛋白、埃博拉病毒包膜蛋白、狂犬病病毒包膜蛋白、杆状病毒包膜蛋白、丙型肝炎病毒包膜蛋白、猫内源反转录病毒包膜蛋白、麻疹病毒包膜蛋白、鼠白血病病毒的兼嗜性4070A和10A1、长臂猿白血病病毒的包膜蛋白、人免疫缺陷病毒的gp120和水疱性口炎病毒的糖蛋白(VSV-G)。
  8. 根据权利要求2所述的方法，其中所述病毒包膜蛋白是水疱性口炎病毒的糖蛋白(VSV-G)。
  9. 根据权利要求2所述的方法，其中所述SB系统中使用的转座酶是SB100X，和/或所述PB系统中使用的转座酶是ePiggyBac。
  10. 根据权利要求2所述的方法，其中所述gag、pol和rev基因、病毒包膜蛋白的编码序列和携带目的核酸片段的病毒基因组转录盒中的一种或多种的转录是受控的。
  11. 根据权利要求10所述的方法，其中所述病毒包膜蛋白是VSV-G并且rev基因和VSV-G的编码序列的转录是受控的。
  12. 根据权利要求10所述的方法，其中所述病毒包膜蛋白是VSV-G并且gag、pol和rev基因以及VSV-G的编码序列的转录是受控的。
  13. 根据权利要求10所述的方法，其中所述受控的转录是通过将所述基因或序列置于诱导表达系统的控制下实现的。
  14. 根据权利要求13所述的方法，其中所述诱导表达系统选自四环素诱导表达系统和Cumate诱导表达系统。
  15. 根据权利要求14所述的方法，其中所述基因或序列被置于Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下。
  16. 根据权利要求15所述的方法，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，和/或VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列、TRE 3GCuO序列或TRE 3GCuO序列-内含子的控制下，和/或VSV-G的转录在TRE advCuO序列、TRE advCuO序列-内含子、 TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子、TRE adv序列-内含子、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下。
  17. 根据权利要求16所述的方法，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列或TRE 3GCuO序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3GCuO序列-内含子的控制下。
  18. 根据权利要求17所述的方法，其中在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在TRE 3GCuO序列-内含子的控制下，并且gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为2-8个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为1:1至4:1。
  19. 根据权利要求18所述的方法，其中gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为4-6个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为2:1至3:1。
  20. 根据权利要求15所述的方法，其中利用SB转座子系统或PB转座子系统将Tet-On反式激活物蛋白的编码序列或Tet-On反式激活物蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列整合在所述宿主细胞的基因组中。
  21. 根据权利要求20所述的方法，其中所述Tet-On反式激活物蛋白是rtTA 3G。
  22. 根据权利要求15所述的方法，其中利用SB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列以及Tet-On反式激活物蛋白的编码序列或Tet-On反式激活物蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用PB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中，或者利用PB转座子系统将慢病毒的gag、pol和rev基因的序列、病毒包膜蛋白的编码序列以及Tet-On反式激活物蛋白的编码序列或Tet-On反式激活物蛋白的编码序列和Cumate操纵子的阻遏物CymR蛋白的编码序列整合在所述宿主细胞的基因组中，然后利用SB转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中。
  23. 通过根据权利要求1-22中任一项所述的方法制备的生产细胞。
  24. 根据权利要求23所述的生产细胞，其中所述生产细胞于2020年4月13日以保藏编号CGMCC No.19675保藏于中国普通微生物菌种保藏管理中心。
  25. 一种用于生产携带目的核酸片段的逆转录病毒载体的生产细胞，所述生产细胞在其基因组中整合有逆转录病毒的gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病毒基因组转录盒序列，所述gag和pol基因的序列、病毒包膜蛋白的编码序列、以及携带目的核酸片段的病 毒基因组转录盒序列的两端分别具有用于被SB转座酶识别的IR/DR序列或用于被PB转座酶识别的ITR序列，并且所述IR/DR序列和所述ITR序列在所述生产细胞中同时存在。
  26. 根据权利要求25所述的生产细胞，其中所述gag和pol基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被SB转座酶识别的IR/DR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被PB转座酶识别的ITR序列，或者所述gag和pol基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被PB转座酶识别的ITR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被SB转座酶识别的IR/DR序列。
  27. 根据权利要求25所述的生产细胞，其中所述逆转录病毒是慢病毒，所述生产细胞在其基因组中还整合有慢病毒的rev基因的序列，并且所述rev基因的序列的两端具有用于被SB转座酶识别的IR/DR序列或用于被PB转座酶识别的ITR序列。
  28. 根据权利要求27所述的生产细胞，其中所述逆转录病毒是HIV-1病毒。
  29. 根据权利要求27所述的生产细胞，其中所述gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被SB转座酶识别的IR/DR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被PB转座酶识别的ITR序列，或者所述gag、pol和rev基因的序列以及病毒包膜蛋白的编码序列的两端分别具有用于被PB转座酶识别的ITR序列，而所述携带目的核酸片段的病毒基因组转录盒序列的两端具有用于被SB转座酶识别的IR/DR序列。
  30. 根据权利要求25所述的生产细胞，其中所述病毒包膜蛋白是水疱性口炎病毒的糖蛋白(VSV-G)。
  31. 根据权利要求23所述的生产细胞或根据权利要求25所述的生产细胞，其中所述目的核酸片段序列两端具有用于位点特异性重组酶系统的识别序列，和/或所述外包膜蛋白的编码序列两端具有用于位点特异性重组酶系统的识别序列。
  32. 用于逆转录病毒载体的目的核酸片段和/或外包膜蛋白替换系统，所述系统包括：根据权利要求31所述的生产细胞，位点特异性重组酶系统，以及待替换的目的核酸片段和/或外包膜蛋白的编码序列。
  33. 根据权利要求32所述的系统，其中所述位点特异性重组酶系统是FLP-FRT或Cre-lox重组酶系统。
  34. 根据权利要求32所述的系统，其中所述生产细胞中原有的目的核酸片段包含标记基因或与标记基因相连和/或所述生产细胞中原有的外包膜蛋白的编码序列与标记基因相连，并且与待替换的目的核酸片段和/或外包膜蛋白的编码序列相连的标记基因与所述生产细胞中原有的标记基因不同。
  35. 根据权利要求34所述的系统，其中所述标记基因是用于真核细胞的抗性基因序列，代谢通路筛选基因，荧光蛋白标记基因，能够用于报告基因检测蛋白酶基因，或其任意组合。
  36. 根据权利要求35所述的系统，其中所述抗性基因序列选自：潮霉素抗性基因、嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、博莱霉素抗性基因。
  37. 根据权利要求35所述的系统，其中所述代谢通路筛选基因选自编码二氢叶酸还原酶(Dihydrofolate reductase)、谷氨酰胺合成酶(Glutamine synthetase)、胸苷激酶(Thymidine kinase)的基因。
  38. 根据权利要求35所述的系统，其中所述荧光蛋白标记基因选自编码绿色荧光蛋白(EGFP)、红色荧光蛋白(dsRed)、樱桃色荧光蛋白(mcherry)、青色荧光蛋白(ECFP)、黄色荧光蛋白(EYFP)、以及荧光蛋白的其它突变衍生蛋白的基因序列。
  39. 根据权利要求35所述的系统，其中所述能够用于报告基因检测蛋白酶基因选自编码萤光素酶(luciferase)、β-半乳糖苷酶(β-galactosidase)、氯霉素乙酰转移酶(chloramphenical acetyltransferase)的基因序列。
  40. 用于替换逆转录病毒的生产细胞中的目的核酸片段和/或外包膜蛋白的方法，所述方法包括：提供根据权利要求31所述的生产细胞，以及利用位点特异性重组酶系统将生产细胞中原有的目的核酸片段和/或外包膜蛋白的编码序列替换为待替换的目的核酸片段和/或外包膜蛋白的编码序列。
  41. 根据权利要求40所述的方法，其中所述位点特异性重组酶系统是FLP-FRT或Cre-lox重组酶系统。
  42. 根据权利要求40所述的方法，其中所述生产细胞中原有的目的核酸片段包含标记基因或与标记基因相连和/或所述生产细胞中原有的外包膜蛋白的编码序列与标记基因相连，并且与待替换的目的核酸片段和/或外包膜蛋白的编码序列相连的标记基因与所述生产细胞中原有的标记基因不同。
  43. 根据权利要求42所述的方法，其中所述标记基因是用于真核细胞的抗性基因序列，代谢通路筛选基因，荧光蛋白标记基因，能够用于报告基因检测蛋白酶基因，或其任意组合。
  44. 根据权利要求43所述的方法，其中所述抗性基因序列选自：潮霉素抗性基因、嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、博莱霉素抗性基因。
  45. 根据权利要求43所述的方法，其中所述代谢通路筛选基因选自编码二氢叶酸还原酶(Dihydrofolate reductase)、谷氨酰胺合成酶(Glutamine synthetase)、胸苷激酶(Thymidine kinase)的基因。
  46. 根据权利要求43所述的方法，其中所述荧光蛋白标记基因选自编码绿色荧光蛋白(EGFP)、红色荧光蛋白(dsRed)、樱桃色荧光蛋白(mcherry)、青色荧光蛋白(ECFP)、黄色荧光蛋白(EYFP)、以及荧光蛋白的其它突变衍生蛋白的基因序列。
  47. 根据权利要求43所述的方法，其中所述能够用于报告基因检测蛋白酶基因选自编码萤光素酶(luciferase)、β-半乳糖苷酶(β-galactosidase)、氯霉素乙酰转移酶(chloramphenical acetyltransferase)的基因序列。
  48. 通过根据权利要求40所述的用于替换逆转录病毒的生产细胞中的目的核酸片段和/或外包膜蛋白的方法获得的逆转录病毒的生产细胞。
  49. 一种用于生产携带目的核酸片段的逆转录病毒载体的方法，所述方法包括以下步骤：
    向根据权利要求23-31中任一项所述的生产细胞或根据权利要求48所述的生产细胞加入诱导表达系统的诱导物，和
    收集和纯化所得的携带目的核酸片段的逆转录病毒载体。
  50. 根据权利要求49所述的方法，其中所述诱导物是四环素或四环素衍生物和/或Cumate及其功能类似物。
  51. 根据权利要求50所述的方法，其中所述四环素衍生物是多西环素。
  52. 通过根据权利要求49所述的方法制备的携带目的核酸片段的逆转录病毒载体。
  53. 根据权利要求23-31中任一项所述的生产细胞、根据权利要求48所述的生产细胞或根据权利要求52所述的携带目的核酸片段的逆转录病毒载体在制备用于向细胞递送目的核酸片段的试剂中的用途。
  54. 一种用于制备用于生产慢病毒载体的包装/生产细胞的方法，所述方法包括将慢病毒的gag、pol和rev基因的序列以及水疱性口炎病毒的糖蛋白(VSV-G)的编码序列转入宿主细胞中，所述gag、pol和rev基因以及VSV-G的编码序列中的一种或多种的转录在Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，和/或VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列、TRE 3GCuO序列或TRE 3GCuO序列-内含子的控制下，和/或VSV-G的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子、TRE adv序列-内含子、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下。
  55. 根据权利要求54所述的方法，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列或TRE 3GCuO序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3GCuO序列-内含子的控制下。
  56. 根据权利要求54所述的方法，其中所述慢病毒的gag、pol和rev基因的序列以及VSV-G的编码序列被整合在宿主细胞的基因组中。
  57. 根据权利要求56所述的方法，其中通过SB转座子系统或PB转座子系统将慢病毒的gag、pol和rev基因的序列以及VSV-G的编码序列整合在宿主细胞的基因组中。
  58. 根据权利要求56所述的方法，其中在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在TRE 3GCuO序列-内含子的控制下，并且gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为2-8 个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为1:1至4:1。
  59. 根据权利要求58所述的方法，其中gag/pol基因在所述宿主细胞的基因组中的插入拷贝数为4-6个拷贝/细胞，并且插入在所述宿主细胞的基因组中的gag/pol与VSV-G的拷贝数之比为2:1至3:1。
  60. 根据权利要求56所述的方法，其中在通过SB转座子系统或PB转座子系统将慢病毒的gag、pol和rev基因的序列以及VSV-G的编码序列整合在宿主细胞的基因组中后，通过相同的转座子系统将携带目的核酸片段的病毒基因组转录盒序列进一步整合在所述宿主细胞的基因组中，或通过瞬时转染的方法将携带目的核酸片段的病毒基因组转录盒序列进一步转入所述宿主细胞中。
  61. 一种用于生产慢病毒载体的包装/生产细胞，所述包装/生产细胞包含慢病毒的gag、pol和rev基因的序列以及水疱性口炎病毒的糖蛋白(VSV-G)的编码序列，所述gag、pol和rev基因以及VSV-G的编码序列中的一种或多种的转录在Tet-On诱导表达系统的单独控制下或Tet-On和Cumate诱导表达系统的双重控制下，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，和/或VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列、TRE 3GCuO序列或TRE 3GCuO序列-内含子的控制下，和/或VSV-G的转录在TRE advCuO序列、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下，和/或gag、pol的转录在真核启动子-内含子、TRE adv序列-内含子、TRE advCuO序列-内含子、TRE 3G序列-内含子或TRE 3GCuO序列-内含子的控制下。
  62. 根据权利要求61所述的用于生产慢病毒载体的包装/生产细胞，其中所述慢病毒的gag基因的序列及/或pol基因的序列及/或rev基因的序列及/或水疱性口炎病毒的糖蛋白(VSV-G)的编码序列被整合在所述包装/生产细胞的基因组中。
  63. 根据权利要求61所述的用于生产慢病毒载体的包装/生产细胞，其中，在Tet-On诱导表达系统单独控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的编码序列的转录在TRE 3G序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3G序列-内含子的控制下；在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列或TRE 3GCuO序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在真核启动子-内含子或TRE 3GCuO序列-内含子的控制下。
  64. 根据权利要求61所述的用于生产慢病毒载体的包装/生产细胞，其中在Tet-On和Cumate诱导表达系统双重控制的情况下，rev的转录在TRE 3G序列的控制下，VSV-G的转录在TRE 3GCuO序列-内含子的控制下，并且gag、pol的转录在TRE 3GCuO序列-内含子的控制下，并且gag/pol基因在所述包装/生产细胞的基因组中的插入拷贝数为2-8个拷贝/细胞，并且插入在所述包装/生产细胞的基因组中的gag/pol与VSV-G的拷贝数之比为1:1至4:1。
  65. 根据权利要求64所述的用于生产慢病毒载体的包装/生产细胞，其中gag/pol基因在所述包装/生产细胞的基因组中的插入拷贝数为4-6个拷贝/细胞，并且插入在所述包装/生产细胞的基因组中的gag/pol与VSV-G的拷贝数之比为2:1至3:1。
  66. 根据权利要求61所述的用于生产慢病毒载体的包装/生产细胞，其于2020年4月13日以保藏编号CGMCC No.19674保藏于中国普通微生物菌种保藏管理中心。
  67. 一种生产慢病毒载体的方法，其包括在适于生产慢病毒载体的条件下培养根据权利要求61至66任一项所述的包装/生产细胞。
PCT/CN2020/115522 2020-04-30 2020-09-16 用于逆转录病毒载体的生产细胞和包装细胞及其制备方法 WO2021218000A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2022564610A JP2023523043A (ja) 2020-04-30 2020-09-16 レトロウイルスベクターの産生細胞およびパッケージング細胞、ならびにそれらの調製方法
US17/766,684 US20230051793A1 (en) 2020-04-30 2020-09-16 Production cell and packaging cell for retroviral vector and preparation method therefor
EP20934080.1A EP4144856A1 (en) 2020-04-30 2020-09-16 Production cell and packaging cell for retroviral vector and preparation method therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010366440.4 2020-04-30
CN202010366440.4A CN113584083A (zh) 2020-04-30 2020-04-30 用于逆转录病毒载体的生产细胞和包装细胞及其制备方法

Publications (1)

Publication Number Publication Date
WO2021218000A1 true WO2021218000A1 (zh) 2021-11-04

Family

ID=78237532

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/115522 WO2021218000A1 (zh) 2020-04-30 2020-09-16 用于逆转录病毒载体的生产细胞和包装细胞及其制备方法

Country Status (5)

Country Link
US (1) US20230051793A1 (zh)
EP (1) EP4144856A1 (zh)
JP (1) JP2023523043A (zh)
CN (1) CN113584083A (zh)
WO (1) WO2021218000A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023147488A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Cytokine associated tumor infiltrating lymphocytes compositions and methods

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115948343A (zh) * 2023-01-30 2023-04-11 吉林大学 表达狂犬病病毒糖蛋白的稳转细胞株及其构建方法与应用
CN116837031B (zh) * 2023-05-29 2024-04-09 皖南医学院第一附属医院（皖南医学院弋矶山医院） 一种生产慢病毒的低背景稳定生产细胞株的构建及其应用
CN116855538A (zh) * 2023-07-11 2023-10-10 梅尔顿(深圳)生物医药技术有限公司 一种复制缺陷型重组病毒扩增用细胞系的制备方法和缺陷性病毒及其应用

Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040510A1 (en) 1997-03-11 1998-09-17 Regents Of The University Of Minnesota Dna-based transposon system for the introduction of nucleic acid into dna of a cell
WO2000075347A2 (en) 1999-06-07 2000-12-14 Wolfgang Hillen Tet repressor-based transcriptional regulatory proteins
US6218185B1 (en) 1996-04-19 2001-04-17 The United States Of America As Represented By The Secretary Of Agriculture Piggybac transposon-based genetic transformation system for insects
WO2002088346A2 (en) 2001-05-01 2002-11-07 National Research Council Of Canada A system for inducible expression in eukaryotic cells
US20020173634A1 (en) 2000-10-31 2002-11-21 Fraser Malcolm J. Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac
WO2003089618A2 (en) 2002-04-22 2003-10-30 Regents Of The University Of Minnesota Transposon system and methods of use
WO2006037215A1 (en) 2004-10-04 2006-04-13 National Research Council Of Canada Expression system, components thereof and methods of use
US7105343B1 (en) 2000-10-31 2006-09-12 University Of Notre Dame Du Lac Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector Piggybac
WO2006122442A1 (en) 2005-05-14 2006-11-23 Fudan University Piggybac as a tool for genetic manipulation and analysis in vertebrates
WO2007058527A2 (en) 2005-11-17 2007-05-24 Academisch Medisch Centrum Inducible expression systems
WO2009003671A2 (en) 2007-07-04 2009-01-08 Max-Delbrück-Centrum für Molekulare Medizin Hyperactive variants of the transposase protein of the transposon system sleeping beauty
WO2010085699A2 (en) 2009-01-23 2010-07-29 The Johns Hopkins University Mammalian piggybac transposon and methods of use
WO2010099296A1 (en) 2009-02-26 2010-09-02 Transposagen Biopharmaceuticals, Inc. Hyperactive piggybac transposases
WO2010099301A2 (en) 2009-02-25 2010-09-02 The Johns Hopkins University Piggybac transposon variants and methods of use
US20100240133A1 (en) 2009-03-20 2010-09-23 The Rockefeller University Compositions and Methods for Transposon Mutagenesis of Human Embryonic Stem Cells
WO2012074758A1 (en) 2010-11-16 2012-06-07 Transposagen Bioharmaceuticals, Inc. Hyperactive piggybac transposases
CN103131672A (zh) * 2011-12-01 2013-06-05 上海市普陀区中心医院 高效表达HIF-1α基因的肠癌细胞株
WO2017046259A1 (en) 2015-09-16 2017-03-23 Ethris Gmbh Improved transposon system for gene delivery
WO2017158029A1 (en) 2016-03-15 2017-09-21 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Enhanced sleeping beauty transposons, kits and methods of transposition
CN108611326A (zh) * 2018-04-24 2018-10-02 高山 一种高效的慢病毒生产体系及其生产方法
WO2018192981A1 (en) * 2017-04-18 2018-10-25 Glaxosmithkline Intellectual Property Development Limited Stable cell lines for retroviral production
CN109312365A (zh) * 2016-02-26 2019-02-05 Ucl商务股份有限公司 用于生产逆转录病毒载体的核酸构建体

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105177031B (zh) * 2015-06-12 2018-04-24 北京艺妙神州医疗科技有限公司 嵌合抗原受体修饰的t细胞及其用途

Patent Citations (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6218185B1 (en) 1996-04-19 2001-04-17 The United States Of America As Represented By The Secretary Of Agriculture Piggybac transposon-based genetic transformation system for insects
US6551825B1 (en) 1996-04-19 2003-04-22 The United States Of America, As Represented By The Secretary Of Agriculture PiggyBac transposon-based genetic transformation system for insects
WO1998040510A1 (en) 1997-03-11 1998-09-17 Regents Of The University Of Minnesota Dna-based transposon system for the introduction of nucleic acid into dna of a cell
WO2000075347A2 (en) 1999-06-07 2000-12-14 Wolfgang Hillen Tet repressor-based transcriptional regulatory proteins
US7105343B1 (en) 2000-10-31 2006-09-12 University Of Notre Dame Du Lac Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector Piggybac
US20020173634A1 (en) 2000-10-31 2002-11-21 Fraser Malcolm J. Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac
WO2002088346A2 (en) 2001-05-01 2002-11-07 National Research Council Of Canada A system for inducible expression in eukaryotic cells
US7745592B2 (en) 2001-05-01 2010-06-29 National Research Council Of Canada Cumate-inducible expression system for eukaryotic cells
WO2003089618A2 (en) 2002-04-22 2003-10-30 Regents Of The University Of Minnesota Transposon system and methods of use
WO2006037215A1 (en) 2004-10-04 2006-04-13 National Research Council Of Canada Expression system, components thereof and methods of use
WO2006122442A1 (en) 2005-05-14 2006-11-23 Fudan University Piggybac as a tool for genetic manipulation and analysis in vertebrates
WO2007058527A2 (en) 2005-11-17 2007-05-24 Academisch Medisch Centrum Inducible expression systems
WO2009003671A2 (en) 2007-07-04 2009-01-08 Max-Delbrück-Centrum für Molekulare Medizin Hyperactive variants of the transposase protein of the transposon system sleeping beauty
WO2010085699A2 (en) 2009-01-23 2010-07-29 The Johns Hopkins University Mammalian piggybac transposon and methods of use
WO2010099301A2 (en) 2009-02-25 2010-09-02 The Johns Hopkins University Piggybac transposon variants and methods of use
WO2010099296A1 (en) 2009-02-26 2010-09-02 Transposagen Biopharmaceuticals, Inc. Hyperactive piggybac transposases
US8592211B2 (en) 2009-03-20 2013-11-26 The Rockefeller University Enhanced PiggyBac transposon and methods for transposon mutagenesis
US20100240133A1 (en) 2009-03-20 2010-09-23 The Rockefeller University Compositions and Methods for Transposon Mutagenesis of Human Embryonic Stem Cells
WO2012074758A1 (en) 2010-11-16 2012-06-07 Transposagen Bioharmaceuticals, Inc. Hyperactive piggybac transposases
CN103131672A (zh) * 2011-12-01 2013-06-05 上海市普陀区中心医院 高效表达HIF-1α基因的肠癌细胞株
WO2017046259A1 (en) 2015-09-16 2017-03-23 Ethris Gmbh Improved transposon system for gene delivery
CN109312365A (zh) * 2016-02-26 2019-02-05 Ucl商务股份有限公司 用于生产逆转录病毒载体的核酸构建体
WO2017158029A1 (en) 2016-03-15 2017-09-21 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Enhanced sleeping beauty transposons, kits and methods of transposition
WO2018192981A1 (en) * 2017-04-18 2018-10-25 Glaxosmithkline Intellectual Property Development Limited Stable cell lines for retroviral production
CN108611326A (zh) * 2018-04-24 2018-10-02 高山 一种高效的慢病毒生产体系及其生产方法

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
AKEF, A. ET AL.: "Splicing promotes the nuclear export of beta-globin mRNA by overcoming nuclear retention elements", RNA, vol. 21, no. 11, 2015, pages 1908 - 1920
GAO QIANG: "ESTABLISHMENT OF A PACKAGING CELL LINE FOR LENTIVIRUS VECTOR", CHINESE MASTER'S THESES FULL-TEXT DATABASE, TIANJIN POLYTECHNIC UNIVERSITY, CN, no. 1, 15 January 2007 (2007-01-15), CN , XP055862237, ISSN: 1674-0246 *
GOSSEN, M.H. BUJARD: "Tight control of gene expression in mammalian cells by tetracycline-responsive promoters", PROC NATL ACAD SCI U S A, vol. 89, no. 12, 1992, pages 5547 - 5551, XP002202393, DOI: 10.1073/pnas.89.12.5547
KAUL, M. ET AL.: "Regulated lentiviral packaging cell line devoid of most viral cis-acting sequences", VIROLOGY, vol. 249, no. 1, 1998, pages 167 - 174, XP002930834, DOI: 10.1006/viro.1998.9327
LACOSTE, A. ET AL.: "An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells", CELL STEM CELL, vol. 5, no. 3, 2009, pages 332 - 342, XP055099142, DOI: 10.1016/j.stem.2009.07.011
LOEW, R. ET AL.: "A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma- retroviral vectors using targeted integration", GENE THER, vol. 17, no. 2, 2010, pages 272 - 280
MICHAEL R. GREEN: "MolecjxLar Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
MULLICK, A. ET AL.: "The cumate gene-switch: a system for regulated expression in mammalian cells", BMC BIOTECHNOL, vol. 6, 2006, pages 43, XP021023579, DOI: 10.1186/1472-6750-6-43
SCHUCHT, R. ET AL.: "A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors", MOL THER, vol. 14, no. 2, 2006, pages 285 - 292, XP005524740, DOI: 10.1016/j.ymthe.2005.12.003
YANT, S. R. ET AL.: "Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase: critical residues for DNA binding and hyperactivity in mammalian cells", MOL CELL BIOL, vol. 24, no. 20, 2004, pages 9239 - 9247, XP002466097, DOI: 10.1128/MCB.24.20.9239-9247.2004
YU, H. ET AL.: "Inducible human immunodeficiency virus type 1 packaging cell lines", J VIROL, vol. 70, no. 7, 1996, pages 4530 - 4537
YUSA, K. ET AL.: "A hyperactive piggyBac transposase for mammalian applications", PROC NATL ACAD SCI USA, vol. 108, no. 4, 2011, pages 1531 - 1536, XP055035951, DOI: 10.1073/pnas.1008322108

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023147488A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Cytokine associated tumor infiltrating lymphocytes compositions and methods

Also Published As

Publication number Publication date
US20230051793A1 (en) 2023-02-16
CN113584083A (zh) 2021-11-02
EP4144856A1 (en) 2023-03-08
JP2023523043A (ja) 2023-06-01

Similar Documents

Publication Publication Date Title
WO2021218000A1 (zh) 用于逆转录病毒载体的生产细胞和包装细胞及其制备方法
EP3380604B1 (en) Stable cell lines for retroviral production
US10450574B2 (en) Transient transfection method for retroviral production
CN110520535B (zh) 用于逆转录病毒产生的稳定细胞系
Sparacio et al. Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors
WO2007133674A2 (en) Lentiviral vector compositions, methods and applications
WO2021232633A1 (zh) 启动元件及包含其的逆转录病毒基因组转录盒和载体
US20050079616A1 (en) Method of transducing ES cells
Tolmachov et al. Designing lentiviral gene vectors
KR20230017799A (ko) 세포 및 유전자 요법을 위한 안정한 바이러스 벡터 생산자 세포를 생산하기 위한 조성물 및 방법
CN116837031B (zh) 一种生产慢病毒的低背景稳定生产细胞株的构建及其应用
KR20230154075A (ko) 세포 및 유전자 치료를 위한 바이러스 벡터 생성자 세포를 생성하고 최적화하기 위한 조성물 및 방법
AU2002351884B2 (en) Method of transducing ES cells
WO2022192261A1 (en) Compositions and methods for producing and characterizing viral vector producer cells for cell and gene therapy
Oliveira Application of functional genomics in the production of viral biopharmaceuticals: Vaccines and Vectors for gene therapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20934080

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022564610

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020934080

Country of ref document: EP

Effective date: 20221130