WO2021232633A1 - Promoter element, and retroviral genome transcription cassette and vector which contain same - Google Patents

Promoter element, and retroviral genome transcription cassette and vector which contain same Download PDF

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WO2021232633A1
WO2021232633A1 PCT/CN2020/115521 CN2020115521W WO2021232633A1 WO 2021232633 A1 WO2021232633 A1 WO 2021232633A1 CN 2020115521 W CN2020115521 W CN 2020115521W WO 2021232633 A1 WO2021232633 A1 WO 2021232633A1
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nucleic acid
vector
plasmid
sequence
transcription cassette
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薛博夫
杨银辉
刘科
马墨
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深圳市深研生物科技有限公司
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
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    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • 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

Definitions

  • the present disclosure relates to the field of viral vectors, in particular, to the field of retroviruses, especially lentiviral vectors; more specifically, to the initiation of transcription in viral genome transcription cassettes that carry target nucleic acid fragments for preparing retroviruses, especially lentiviral vectors. Elements, and viral genome transcription cassettes and vectors containing this element.
  • 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 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).
  • FIV Feline immunodeficiency virus
  • BIV Bovine immunodeficiency virus
  • CAEV Caprine arthritis-encephalitis virus
  • a vector based solely on Equine infectious anemia virus (EIAV) has been developed to the clinical use stage (ProSavin) for the treatment of Parkinson's.
  • 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, and its 5'-end LTR is replaced by the cytomegalovirus early promoter, and the 3'-LTR is replaced by the simian virus-40 polyadenylic acid (SV40polyA) sequence, and the HIV-1 Envelope gene env.
  • 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 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 (M
  • 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, Hela cells, CHO cells, BHK cells, COS cells, NIH/3T3 cells, Vero cells, HT1080 cells, Te671 cells, CEM Cells, NSO 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 aforementioned nucleic acid fragment such as a plasmid
  • retroviral vectors Most of the retroviral vectors currently in use are derived from Moloney Murine Leukemia Virus (Mo-MLV), and this vector system has achieved many design improvements over the years.
  • the rapid and efficient method of preparing retroviral vectors is by transiently transfecting 293-based packaging cell lines, such as Phoenix-ECO and Phoenix-AMPHO (Dr. Gary P. Nolan, Stanford University) or other commercially available packaging cell lines such as Plat. -A, Plat-E, and Plat-GP (Cell Biolabs); AmpphoPak-293; EcoPak2-293; and RetroPack PT67 (Clonteck).
  • 293-based packaging cell lines such as Phoenix-ECO and Phoenix-AMPHO (Dr. Gary P. Nolan, Stanford University) or other commercially available packaging cell lines such as Plat. -A, Plat-E, and Plat-GP (Cell Biolabs); AmpphoPak-293; EcoPak2-293; and RetroPack PT67 (Clonteck).
  • the retroviral vector In order to pseudotype the retroviral vector with other envelope proteins (such as vesicular stomatitis virus glycoprotein (VSV-G)), it can be used in packaging cell lines that only express Mo-MLV gag and pol genes (such as Plat- GP, Cell Biolabs; GP2-293, Clontech) were simultaneously transfected with a transfer vector plasmid and a plasmid expressing the target envelope protein; or by combining a plasmid expressing Mo-MLV gag and pol genes (such as pUMVC-plasmid, Addgene, #8449 ), the plasmid expressing the target envelope protein and the transfer vector plasmid are transiently transfected into 293 cells at the same time.
  • VSV-G vesicular stomatitis virus glycoprotein
  • 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. , So as to establish a packaging cell line. Afterwards, 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.
  • the present disclosure significantly increases the production titer of the retrovirus/lentiviral vector by constructing a Tet-responsive element (TRE) that uses the Tet-On inducible expression system to regulate the transcription of a retroviral/lentiviral genome transcription cassette carrying a target nucleic acid fragment.
  • TRE Tet-responsive element
  • 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 self-LTR sequence or a chimeric promoter constructed by other heterologous promoters and LTR), 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 (for example, constructing the above transcription cassette into a transfer vector plasmid or a viral vector) is delivered to the host cell, Through the transcriptional molecular mechanism of host cells, it is transcribed into corresponding viral genome RNA fragments that can be packaged into viral vectors and carry target nucleic acid fragments.
  • the retroviral genome transcription cassette carrying the target nucleic acid fragment needs to provide the cis-acting elements required for transcription, packaging and transduction, mainly including: (1) Long terminal repeats (LTR) fragments: the main function of LTR It regulates the reverse transcription of viral genome from RNA to DNA, DNA provirus integration in the host cell genome and transcription of viral mRNA fragments after integration.
  • LTR Long terminal repeats
  • LTR is composed of three functional domains of U3 (unique-3'), R (repeat) and U5 (unique-5') according to the structure of U3-R-U5, and the U3 functional domain mainly provides transcription promoter and enhancer functions;
  • the R functional domain participates in the reverse transcription process and encodes the 5'capping sequence (5'cap) and polyadenylation (polyA) signal of the viral RNA genome;
  • the U5 functional domain is the start site of reverse transcription.
  • the currently commonly used SIN vector deletes the U3 functional domain in the 3'LTR, and regulates the transcription of the entire viral RNA genome carrying the target nucleic acid fragment through a chimeric promoter composed of an exogenous promoter and the R-U5 functional domain in the 5'LTR Transcription of the box.
  • PBS Primer binding site
  • tRNA specific transfer RNA
  • ⁇ packaging signal Located between the primer binding site and the gag open reading frame, it is a necessary element for the packaging of viral RNA genome into viral particles.
  • PPT Polypurine tract
  • Rev response element binds to the regulatory protein Rev encoded by the lentivirus, which is essential for the transport of unspliced and incompletely spliced viral mRNA from the nucleus to the cytoplasm after transcription.
  • the inclusion of the RRE sequence in the lentiviral genome transcription cassette can significantly improve the packaging efficiency of the lentiviral vector.
  • the 3'end of the target nucleic acid fragment in the genome transcription cassette of the retroviral/lentiviral vector is connected with posttranscriptional regulatory elements (posttranscriptional regulatory elements) can significantly increase the expression level of the transgene in the target nucleic acid fragment.
  • the post-transcriptional regulatory element (Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element, WPRE) or the Constitutive Transport Element (CTE) of Mason-Pfizer monkey virus (Mason-Pfizer monkey virus).
  • WPRE Woodchuck Hepatitis Virus
  • CTE Constitutive Transport Element
  • vector refers to a nucleic acid molecule, which is usually used as a vehicle for artificially carrying foreign genetic material, such as the above-mentioned nucleic acid fragment of interest, into another cell, where it is replicated and/or expressed. Functionally, all vectors can generally be used to clone and carry foreign nucleic acid fragments of interest, and there are also specially designed expression vectors for nucleic acid fragment transcription and protein expression. Plasmids, viral vectors, cosmids and artificial chromosomes are the four main types of vectors. Among them, the most commonly used vector is a plasmid.
  • All engineered plasmid vectors contain an origin of replication in bacteria, a multiple cloning site for inserting target nucleic acid fragments, and a marker gene for selecting positive strains.
  • Viral vectors are another commonly used vector, usually used to deliver genetic material, such as nucleic acid fragments of interest, into cells. This process can be performed in vivo (in vivo) or in cell culture (in vitro).
  • a variety of molecular mechanisms based on the evolution of the virus itself such as the protection of genetic material, the selection of host cells based on the receptor, the delivery of genetic material into the host cell, the replication and/or expression in the host cell, the protection of the host cell
  • the adjustment of growth, metabolism, reproduction and defense mechanisms, as well as the suppression and/or escape of the immune system in higher animals, can effectively transfer target nucleic acid fragments to infected target cells.
  • viral vectors are often used in gene therapy, cell therapy, immunotherapy, and vaccine development.
  • the gene fragments used for packaging retrovirus/lentivirus and the viral genome transcription cassette carrying the target nucleic acid fragment can be produced by constructing the above-mentioned various vectors and transferring them into host cells for viral vector production.
  • an inducible expression system is used to control the transcription of a viral genome transcription cassette carrying a target nucleic acid fragment into a host cell.
  • examples of usable inducible expression systems include, but are not limited to, the Tet-off system (see, for example, Gossen, M. and H. Bujard (1992). "Tight control of gene expression in mammalian cells by tetracycline -responsive promoters.”Proc Natl Acad Sci USA 89(12):5547-5551; Yu,H.,et al.(1996).”Inducible human immunodeficiency virus type 1 packaging cell lines.”J Virol 70(7):4530 -4537; Kaul, M., et al. (1998).
  • 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: 13-756bp in SEQ ID NO: 11) and TRE adv ) and the third generation Tet-On inducible expression system (Tet-On 3G, Clontech, in which the transactivator rtTA and the response element TRE are respectively referred to as rtTA 3G hereinafter (Encoding nucleic acid sequence: 13-756bp in SEQ ID NO: 10) and TRE 3G ).
  • the transactivators rtTA and TRE in the second and third generation Tet-On inducible expression systems can be used in combination.
  • rtTA adv can be used in combination with TRE 3G
  • rtTA 3G can also be used in combination with TRE adv .
  • the present disclosure relates to the regulation of transcription cassettes of viral genomes carrying target nucleic acid fragments based on tetracycline-dependent nucleic acid sequences (Tet response elements, TRE) during the production process of retroviral/lentiviral vectors.
  • the Tet response element TRE includes at least 2 copies of a TetO-operator sequence (TetO) that binds to a tetracycline-dependent transactivator (reverse tetracycline controlled transactivator, rtTA), and 1 copy of a TATA
  • TetO TetO-operator sequence
  • rtTA reverse tetracycline controlled transactivator
  • the minimal promoter sequence of the box sequence preferably, the TRE sequence is shown in SEQ ID NO: 17 and SEQ ID NO: 18.
  • the chimeric promoter that regulates the transcription of the viral genome transcription cassette carrying the nucleic acid fragment of interest includes the above-mentioned Tet response element TRE and the R-U5 functional domain in the LTR sequence of the retrovirus/lentivirus; preferably, The R-U5 functional domain is connected downstream of the TATA box sequence in the Tet response element and is separated from it by 20-24 base pairs, more preferably 24 base pairs. In one aspect of the present disclosure, the R-U5 functional domain is the R-U5 functional domain of the HIV-1 lentivirus.
  • the Tet response element and the R-U5 functional domain of the HIV-1 lentivirus are embedded
  • the combined response elements are TRE1-RU5 (as shown in SEQ ID NO: 19) and TRE2-RU5 (as shown in SEQ ID NO: 20).
  • the ⁇ -retroviral genome transcription cassette carrying the target nucleic acid fragment can be derived from the transfer vector plasmid of the MSCV (Murine Stem Cell Virus) retroviral expression system using 5'LTR as the promoter
  • MSCV Human Stem Cell Virus
  • 5'LTR as the promoter
  • MoMLV Moloney Murine Leukemia
  • pBABE-hygro-hTERT Additional Murine Leukemia
  • pBABE-puro Additional reverse transcription expression systems based on CMV-R-U5 embedded
  • 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 cassettes in the above-mentioned lentiviral transfer vector plasmids are in principle applicable to the method of preparing viral vectors through the viral genome transcription cassettes carrying the target nucleic acid fragments regulated by Tet response elements 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.
  • 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
  • Retroviral/lentiviral vectors can be prepared by introducing gag genes, pol genes, rev genes (for lentiviral vectors), envelope glycoprotein genes, and transcription regulation by Tet response elements in the host cell.
  • the viral genome transcription cassette carrying the target nucleic acid fragment (at the same time needs to be transferred to the transactivator rtTA that regulates the Tet response element).
  • the above-mentioned retroviral/lentiviral vector production method can be realized by "transient production", which is expressed as a viral genome transcription cassette (hereinafter referred to as viral genome transcription) containing a target nucleic acid fragment regulated by Tet response element transcription as described in the present disclosure.
  • vectors such as plasmids or viral vectors
  • viral vectors are prepared without integration into the host cell genome.
  • a vector construct (such as a plasmid Or viral vectors) are all transformed into host cells by transient transfection to prepare retroviral/lentiviral vectors.
  • the transactivator rtTA coding sequence is first stably integrated into the genome of the host cell, so that the host cell stably expresses the rtTA protein.
  • a vector construct such as a plasmid or a viral vector
  • the gag gene, pol gene, rev gene for lentiviral vectors
  • envelope glycoprotein gene and viral genome transcription cassette All were transformed into host cells by transient transfection to prepare retroviral/lentiviral vectors.
  • the transactivator rtTA coding sequence is first stably integrated into the genome of the host cell, and at the same time one of the gag gene, pol gene, rev gene (for lentiviral vectors), and envelope glycoprotein gene is integrated.
  • Species multiple or all of them are stably integrated into the genome of the host cell to construct a packaging cell line, and then the gag gene, pol gene, rev gene (for lentiviral vector) and envelope glycoprotein that are not stably integrated in the packaging cell Genes in the form of vector constructs (such as plasmids or viral vectors) and the vector constructs of viral genome transcription cassettes (such as plasmids or viral vectors) are simultaneously transferred into the above-mentioned packaging cells by transient transfection to prepare retrovirus/lentiviral vectors .
  • vector constructs such as plasmids or viral vectors
  • viral genome transcription cassettes such as plasmids or viral vectors
  • the transactivator rtTA coding sequence, gag gene, pol gene, rev gene (for lentiviral vector), and envelope glycoprotein gene are all stably integrated into the genome of the host cell to construct a packaging cell.
  • the viral genome transcription cassette is transformed into the above-mentioned packaging cell by transient transfection method to prepare a retrovirus/lentiviral vector.
  • tetracycline or its derivatives need to be added to induce the preparation of retroviral/lentiviral vectors.
  • retroviral/lentiviral vector production method can also be achieved by "stable production", which is represented by the expression of a viral genome transcription cassette (hereinafter referred to as viral genome) containing a target nucleic acid fragment regulated by the Tet response element in the present disclosure.
  • Transcription cassette) together with packaging genes such as gag gene, pol gene, rev gene (for lentiviral vectors), envelope glycoprotein gene and transactivator rtTA coding sequence are all stably integrated into the genome of the host cell to construct a production cell line, And by adding tetracycline or its derivatives to induce preparation of retroviral/lentiviral vectors.
  • the above-mentioned packaging cells and production cells for constructing retroviral/lentiviral vectors can be transposed by a transposon system such as Sleeping Beauty (SB) transposon system and/or PiggyBac (PB) Subsystem implementation.
  • SB Sleeping Beauty
  • PB PiggyBac
  • host cells that can be used to prepare retroviral/lentiviral vectors are mammalian cells.
  • host cells suitable for use in the present disclosure are 293T cells, HepG2 cells, Hela 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.
  • host cells can be cultured adherently or in suspension.
  • the host cell can be cultured with or without addition of serum.
  • 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 10-6M; preferably, its binding constant Ka reaches or is stronger than 10-9M.
  • the tetracycline derivative may be selected from, for example, doxycycline (Dox), anhydrotetracycline (Atc), chlortetracycline, oxytetracycline, and deoxytetracycline.
  • the present disclosure provides the following:
  • Item 1 Nucleic acid sequence, which contains the response element TRE of the Tet-On system and the R-U5 functional domain in the retroviral long terminal repeat (LTR).
  • LTR retroviral long terminal repeat
  • Item 2 The nucleic acid sequence of item 1, wherein the R-U5 functional domain is downstream of the TATA box of the TRE and separated from it by 15-30 bp.
  • Item 3 The nucleic acid sequence of item 2, wherein the R-U5 functional domain is downstream of the TATA box of the TRE and separated from it by 24 bp.
  • Item 4 The nucleic acid sequence of item 1, wherein the sequence of TRE is shown in SEQ ID NO: 17 or SEQ ID NO: 18.
  • Item 5 The nucleic acid sequence of item 1, whose sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 20.
  • a retroviral genome transcription cassette which contains the Tet-On system response element TRE or the nucleic acid sequence of any one of items 1-5 for controlling the transcription of the transcription cassette, located in the Tet-On system
  • the response element TRE or the cis-acting element used for retrovirus packaging downstream of the nucleic acid sequence of any one of items 1-5 and a multiple cloning site for inserting the target nucleic acid fragment.
  • Item 7 The retroviral genome transcription cassette of Item 6, wherein the cis-acting element includes a long terminal repeat (LTR), a primer binding site (PBS) and a viral packaging signal (phi signal).
  • LTR long terminal repeat
  • PBS primer binding site
  • phi signal viral packaging signal
  • Item 8 The retroviral genome transcription cassette of item 6, wherein the retrovirus is a lentivirus.
  • Item 9 The retroviral genome transcription cassette of Item 8, wherein the cis-acting element includes a long terminal repeat (LTR), a primer binding site (PBS), a viral packaging signal (phi signal), and a central polypurine Bundle (cPPT) and rev protein response element (RRE).
  • LTR long terminal repeat
  • PBS primer binding site
  • phi signal viral packaging signal
  • RRE rev protein response element
  • Item 10 The retroviral genome transcription cassette of Item 8, wherein the cis-acting element further includes a woodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE).
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory sequence
  • Item 11 The retroviral genome transcription cassette of item 7 or 9, wherein the long terminal repeat sequence is a wild-type U3-R-U5 sequence capable of self-replication or a self-suppressive SIN sequence with U3 sequence deleted .
  • Retroviral genome transcription cassette which is obtained by inserting a nucleic acid fragment of interest into the multiple cloning site of the retroviral genome transcription cassette of any one of items 6-11.
  • Item 13 A vector comprising the nucleic acid sequence of any one of items 1-5 or the retroviral genome transcription cassette of any one of items 6-12.
  • Item 14 The vector of item 13, which is a plasmid vector or a viral vector.
  • Item 15 A host cell comprising the nucleic acid sequence of any one of items 1-5, the retroviral genome transcription cassette of any one of items 6-12, or the 13th or 14th item Carrier.
  • Item 16 The nucleic acid sequence of any one of items 1-5, the retroviral genome transcription cassette of any one of items 6-12, the vector of item 13 or 14, or the host of item 15 Cells are used to produce retroviral vectors carrying target nucleic acid fragments.
  • Item 17 The use of item 16, wherein the nucleic acid sequence of any one of items 1-5, the retroviral genome transcription cassette of any one of items 6-12, and the vector of item 13 or 14 , Or the host cell of item 15 is used for transient or stable production of the retroviral vector carrying the nucleic acid fragment of interest.
  • Figure 1 is a schematic diagram of the actual part of the plasmid in this disclosure.
  • Figure 2 shows the comparison of the toxin production titer of each transfer vector plasmid in 293T cells by transient transfection method according to an embodiment.
  • the abscissa is the combination of each transfer vector plasmid and the rtTA expression plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
  • Figure 3 shows the comparison of the toxin production titer of each transfer vector plasmid by transient transfection method in 293T cells stably expressing rtTA according to an embodiment.
  • the abscissa is the chimeric promoter or chimeric response element used in each transfer vector plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
  • Figure 4 shows the comparison of the toxin production titer of each transfer vector plasmid in EuLV293T packaging cells by transient transfection method according to an embodiment.
  • the abscissa is the chimeric promoter or chimeric response element used in each transfer vector plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
  • Figure 5 shows the effect of various promoters or response elements that regulate transcription of viral genome transcription cassettes on toxin production titer in a stable production cell of a lentivirus according to an embodiment.
  • the abscissa is the chimeric promoter or chimeric response element used to transcribe the viral genome transcription cassette in each lentivirus stable production cell line, and the ordinate is the RLU value of the Luciferase experiment for detecting 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, ligation and ligation of two or more DNA fragments by T4 DNA ligase
  • the methods for product-competent cell transformation, plasmid preparation and identification in small quantities 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-10Gold, Hunan Fenghui Biotechnology Co., Ltd., JZ011
  • SEQ ID NO:1 to SEQ ID NO:16 shown by the nucleic acid sequence GenScript was synthesized 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 the plasmid construction
  • Table 2 is the description of the composition of the elements of the sequence SEQ ID NO: 1 to SEQ ID NO: 20
  • 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 by different codons, mutants of the above functional element sequences and gene sequences, and various functions 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 sequences SEQ ID NO: 2 (2900bp) and SEQ ID NO: 3 (1386bp) were digested with NotI and AsiSI, and they were connected to plasmid 18BF003 (SEQ ID NO: 1,1893bp). The NotI and AsiSI restriction sites were used 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-BGI fragment to construct plasmid 18BF063.
  • Plasmid 18BF068 use pMD2.G (Addgene, #12259) as template, C-VSVG-F (SEQ ID NO: 21) and C-VSVG-R (SEQ ID NO: 22) as primers for PCR amplification of VSV -G gene fragment (1565bp), then cut with ClaI and XhoI and ligated to the ClaI and XhoI sites of 18BF063 plasmid to construct plasmid 18BF068.
  • plasmids 18BF074 and 19BF126 use pMDLg/pRRE (Addgene, #12251) as template, C-RRE-F (SEQ ID NO: 25) and C-RRE-R (SEQ ID NO: 26) as primers for PCR amplification Increase the RRE gene fragment (400bp); C-GagPol-F (SEQ ID NO: 27) and C-GagPol-R (SEQ ID NO: 28) are primers for PCR amplification of the gag/pol gene fragment (4336bp), and then use XbaI The two DNA fragments were digested with XhoI and EcoRI and XbaI respectively and ligated to the EcoRI and XhoI digestion sites of the 18BF007 plasmid to construct the 18BF074 plasmid. The 18BF074 plasmid was digested with BstBI, the 8758bp (18BF074) fragment was recovered from the gel and ligated with T4
  • the synthetic sequence SEQ ID NO: 9 (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 sequences SEQ ID NO: 10 (768 bp) and SEQ ID NO: 11 (765 bp) 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: 8 (1496 bp) 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 18BF085 plasmid to construct the 19BF075 plasmid.
  • the synthetic sequences SEQ ID NO: 14 (1806 bp) and SEQ ID NO: 12 (1320 bp) were digested with BamHI and XhoI, and XhoI and BglII, respectively, and connected to the BamHI and BglII digestion sites of the 18BF011 plasmid to construct the 18BF031 plasmid.
  • plasmid 19BF081 use pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252) as template, hPGK-F (SEQ ID NO: 29) and hPGK-R (SEQ ID NO: 30) as primers for PCR amplification Increase the PGK gene fragment (706bp); use pGL3-Basic (Promega, E1751) as a template, and Luc-F (SEQ ID NO: 31) and Luc-R (SEQ ID NO: 32) as primers to amplify the luciferase gene fragment ( 1728bp), after that, the two DNA fragments were digested with MluI and BamHI (538bp fragments recovered from the gel) and BamHI and XhoI, respectively, and connected to the MluI and XhoI sites of the 19BF126 plasmid, replacing the original plasmid DNA sequence to construct 18YYH26 Plasmid.
  • the synthetic sequence SEQ ID NO: 15 (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: 16 (1320 bp) was digested with XhoI and BglII and connected to the XhoI and BamHI sites of the 19BF080 plasmid to construct the 19BF214 plasmid.
  • Plasmid 18YYH26 was digested with PacI and XhoI, and the 2272bp DNA fragment was recovered by gel respectively, and the recovered fragment was ligated to the PacI and XhoI restriction sites of the 19BF214 plasmid to construct the 19BF081 plasmid.
  • Plasmid 19BF081 was digested with PacI and PvuII, and the 4224bp gene fragment was recovered from the gel and ligated to the PacI and PvuII sites of plasmids 19BF120, 19BF121 and 19BF122 respectively to construct 19BF123, 19BF124 and 19BF125 plasmids, respectively.
  • Example 2 Preparation of a lentiviral vector by transiently transfecting a Tet response element-regulated viral genome transcription cassette construct in 293T cells
  • the coding sequence of the transactivator rtTA adv or rtTA 3G of the transactivator rtTA adv or rtTA 3G of the expression system is induced by the genes required for lentivirus packaging, rev, VSV-G, gag/pol and Tet-On, and regulated by the Tet response element described in the present disclosure.
  • the method of transiently transfecting 293T cells with the construct of the viral genome transcription cassette carrying the target nucleic acid fragment produces lentivirus, and compares the viral genome transcription cassette regulated by the Tet response element described in the present disclosure and contrasts the use of constitutive activity
  • the toxin production titer of the viral genome transcription cassette regulated by the promoter produces lentivirus, and compares the viral genome transcription cassette regulated by the Tet response element described in the present disclosure and contrasts the use of constitutive activity.
  • This example verifies 4 kinds of transfer vector plasmids 19BF081 (constitutively active promoter RSV-RU5), 19BF123 (constitutively active promoter CMV-RU5), 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5), respectively
  • the RSV or CMV constitutively active promoter, or the TRE1 or TRE2Tet response element described in the present disclosure is used to regulate the transcription of the lentiviral genome expression cassette carrying the transgene of interest.
  • the hPGK-Luciferase-IRES-EGFP sequence was used as the target nucleic acid fragment, and the toxin production titer of each transfer vector plasmid under transient production conditions was compared based on the Luciferase enzyme activity.
  • the specific experimental method is as follows.
  • the molar ratio of plasmid pMD2.G (Addgene 12259): pMDLg/PRRE (Addgene 12251): pRSV-Rev (Addgene 12253): rtTA (19BF257 or 19BF256): the transfer vector plasmid is 1:1:1:1:1.
  • the transfer vectors are 19BF081, 19BF123, 19BF124, and 19BF125; the Tet-On transactivator encoding plasmid is 19BF257 (rtTA 3G ) or 19BF256 (rtTA adv ), and multiple wells are set for each sample.
  • the HT1080 cell Luciferase virus titer detection method was used to detect the toxin production titer of each sample.
  • the specific operation is as follows: 24 hours after the induction of toxin production, HT1080 cells were seeded in a 96-well plate (Corning 3916) with 1E+04 cells per well , The medium is DMEM complete medium. One hour before the detection of titer, the medium of HT1080 cells was replaced with DMEM complete medium containing 8 ⁇ g/ml polybrene (Sigam, H9268).
  • the virus supernatant obtained after the above centrifugation was added to a 96-well plate with 50 ⁇ l of each sample in three wells, and 50 ⁇ L of DMEM complete medium was added to the negative control well.
  • DMEM fetal calf serum
  • the detection instrument is a fluorescence microplate reader (Perkin Elmer Victor V). The test results are shown in Figure 1.
  • Figure 2 shows that when the lentivirus is packaged by transiently transfecting 293T cells, the TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) Tet chimeric response elements are used to regulate the transcription of the transfer vector plasmid of the lentiviral genome expression cassette.
  • the toxin production titer is significantly higher than that of traditional transfer vector plasmids using constitutively active promoters.
  • the toxin production titer is 2.93 times or 2.33 times that of RSV-RU5 or CMV-RU5 , respectively; when matched with rtTA 3G , it produces toxin drops The degree is 2.55 times or 2.02 times that of RSV-RU5 or CMV-RU5, respectively.
  • the toxin production titers are 1.75 times or 1.39 times that of RSV-RU5 or CMV-RU5 , respectively; when matched with rtTA 3G , the toxin production drops The degree is 1.83 times or 1.45 times that of RSV-RU5 or CMV-RU5, respectively.
  • Example 3 Preparation of a lentiviral vector by transiently transfecting a Tet response element-regulated viral genome transcription cassette construct in 293T cells stably expressing rtTA
  • the transfer vector plasmid used in this example is the same as that described in Example 2, and they are 19BF081, 19BF123, 19BF124, and 19BF125, respectively.
  • the specific experimental method is as follows.
  • Two cell lines, 293T-rtTA adv and 293T-rtTA 3G were seeded in a 24-well plate with 1.5E+05 cells per well, and the culture volume was 500 ⁇ L DMEM complete medium. After 24 hours of inoculation, cells were transfected according to the PEI method. The operation method is as follows: during transfection, add 50 ⁇ L of transfection reagent to each well, which contains 0.8 ⁇ g of total plasmid and 3.2 ⁇ g of PEI MAX (Polysciences, 24765-1).
  • the molar ratio pMD2.G (Addgene 12259): pMDLg/PRRE (Addgene 12251): pRSV-Rev (Addgene 12253): transfer vector plasmid is 1:1:1:1.
  • the transfer vector is 19BF081 (RSV-RU5), 19BF123 (CMV-RU5), 19BF124 (TRE1-RU5) or 19BF125 (TRE2-RU5), and multiple holes are set for each sample. After 3 hours of transfection, the medium was changed, and an inducer (2mmol/L sodium butyrate, 1 ⁇ g/ml DOX) was added to one of the multiple wells of each sample, and only 2mmol/L sodium butyrate was added to the other multiple well.
  • an inducer (2mmol/L sodium butyrate, 1 ⁇ g/ml DOX
  • the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes.
  • the Luciferase virus titer of HT1080 cells was detected, and the detection method was the same as that in Example 2, and the result is shown in FIG. 3.
  • the induced toxin production titers of 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5) were 1.57E+06RLU and 1.38E+06RLU, respectively; they were 19BF081 (RSV-RU5) transfer vector plasmid 6.7 times and 5.9 times of the transfer vector plasmid of 19BF123 (CMV-RU5), 2.48 times and 2.18 times, respectively.
  • the induced toxin production titers of 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5) were 2.36E+06RLU and 1.97E+6RLU, respectively; they were 19BF081 (RSV-RU5) transfer vector plasmids, respectively 7.7 times and 6.5 times, respectively, 2.4 times and 2.0 times of the transfer vector plasmid of 19BF123 (CMV-RU5).
  • Example 4 Preparation of a lentiviral vector by transiently transfecting a Tet response element-regulated viral genome transcription cassette construct in EuLV293T packaging cells stably expressing lentiviral packaging genes
  • the "Sleeping Beauty (SB)" transposon system was first constructed to stably integrate the genes rev, VSV-G and gag/pol required for lentivirus packaging in the 293T genome, as well as the Tet-On inducible expression system
  • the expression of rev gene and VSV-G gene is regulated by the Tet-On and Cumate compound induction expression system.
  • the plasmids used to construct the cell line are 18BF072 and 18BF068; the expression of gag/pol gene is regulated by the CMV promoter and is used to construct cells
  • the plasmid of the line is 18BF074; the expression of the transactivator rtTA 3G and the repressor CymR protein are regulated by the CAGGS promoter and CMV promoter, respectively, and the expression cassettes of the two proteins are jointly constructed on the 19BF075 plasmid.
  • the construct (transfer vector plasmid) of the viral genome transcription cassette carrying the target nucleic acid fragment regulated by the Tet response element of the present disclosure was transiently transfected into the above-mentioned EuLV293T packaging cell, and compared with the regulation of the Tet response element of the present disclosure
  • the viral genome transcription cassette was compared with the toxin production titer of the viral genome transcription cassette regulated by a constitutively active promoter.
  • the transfer vector plasmid used in this example is the same as that described in Example 2, and they are 19BF081, 19BF123, 19BF124, and 19BF125, respectively.
  • the specific experimental methods are as follows:
  • Example 2 and Example 3 for the experimental procedures of cell culture, PEI transfection, and hygromycin screening. Inoculate 293T cells (ATCC, CRL3216) according to 1.5E+06 cells in a 60mm culture dish with 3ml DMEM complete medium (DMEM (Sigma, D6429) supplemented with 10% FBS (ExCell, 11H116)) at 37°C Cultivate for 24 hours under the condition of 5% CO 2.
  • DMEM complete medium DMEM (Sigma, D6429) supplemented with 10% FBS (ExCell, 11H116)
  • Plasmid transfection was carried out by the 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 19BF075:19BF72:18BF068:19BF74:18BF019 was 3:3:2:12: 2 Perform transfection. Twenty-four hours after transfection, all cells were inoculated into a 100mm petri dish and continued to be cultured, and 200 ⁇ g/ml hygromycin (Sangong A600230-0001) was added for selection. Maintain this selection pressure and subculture until the cells grow stably.
  • EuLV293T cells were cultured at 37°C and 5% CO 2 in a DMEM (Sigam, D6429) medium containing 10% FBS (ExCell, 11H116).
  • the cell line was seeded in a 24-well plate with 1.5E+05 cells per well, and the culture volume was 500 ⁇ L.
  • 50 ⁇ L of transfection reagent was added to each well, which contained 0.26 ⁇ g of transfer vector plasmid, 0.54 ⁇ g of 18BF003 plasmid and 3.2 ⁇ g of PEI MAX (Polysciences, 24765-1), and each condition was equipped with multiple wells.
  • the induced toxin production titers of TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) were 2.42E+06RLU and 2.14E+06RLU, respectively; they were 3.4 times and 3.0 times that of the transfer vector plasmid of 19BF081 (RSV-RU5), respectively It is 1.6 times and 1.4 times of the transfer vector plasmid of 19BF123 (CMV-RU5).
  • RSV-RU5 transfer vector plasmid of 19BF081
  • CMV-RU5 transfer vector plasmid of 19BF123
  • Example 5 Preparation of lentivirus in a stable production cell line of lentivirus by a viral genome transcription cassette regulated by Tet response elements
  • the "PiggyBac (PB) transposon system derived from Trichoplusia ni) was used to transcribe viral genomes carrying hPGK-Luciferase-EGFP target nucleic acid fragments that are regulated by different promoters or Tet response elements.
  • the cassette is stably integrated into the genome of the EuLV293T packaging cell prepared in Example 4.
  • the transfer vector plasmids used to construct the lentivirus production cell line are: 19BF081, 19BF123, 19BF124 and 19BF125.
  • the lentivirus is prepared by induction.
  • the specific experimental methods are as follows:
  • EuLV293T cells were seeded in a 60mm culture dish with 1.5E+06 cells per well.
  • the medium was 3ml DMEM complete medium.
  • transfection was carried out according to the PEI method.
  • the total plasmid The amount is 5.5ug, of which the transfer vector plasmid: 18BF031 (expressing PB transposase) molar ratio is 10:1 for transfection, and the total amount of PEI is 22ug.
  • the transfer vector plasmid is 19BF081, 19BF123, 19BF124 or 19BF125.
  • the above-mentioned four lentivirus stable production cells were seeded in a 24-well plate with 1.5E+05 cells per well, and the culture volume was 500 ⁇ L, and each cell was set to replicate wells. After culturing for 24 hours, change the fresh medium and add inducer (5mmol/L sodium butyrate, 1 ⁇ g/ml DOX and 200 ⁇ g/ml Cumate) to one of the multiple wells to set the induction group, and add only the same amount of butyl to the multiple wells. Sodium is set as the non-induced group. After culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes. The Luciferase virus titer of HT1080 cells was detected, and the detection method was the same as that in Example 2, and the result is shown in FIG. 5.
  • inducer 5mmol/L sodium butyrate, 1 ⁇ g/ml DOX and 200 ⁇ g/ml Cumate
  • the induced toxin production titers of EuLV293T-19BF124 (TRE1-RU5) and EuLV293T-19BF125 (TRE2-RU5) production cell lines were 3.64E+07RLU and 2.37E+07RLU, respectively; EuLV293T-19BF081 (RSV-RU5) production cells, respectively 9.1 times and 5.9 times of the induced toxin production titers of EuLV293T-19BF123 (CMV-RU5); 2.46 times and 1.61 times of the induced toxin production titers of EuLV293T-19BF123 (CMV-RU5) production cells, respectively. Under non-induced conditions, no virus titer can be detected in all lentivirus stable production cell lines.

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Abstract

On the basis of a response element TRE or TRE of a Tet-On system and an activation element of an R-U5 functional domain in a long terminal repeat (LTR), provided are a retroviral/lentiviral genome transcription cassette and vector containing the promoter element.

Description

启动元件及包含其的逆转录病毒基因组转录盒和载体Promoter element and retroviral genome transcription cassette and vector containing it 技术领域Technical field
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，涉及用于制备逆转录病毒尤其是慢病毒载体的携带目的核酸片段的病毒基因组转录盒中启动转录的元件，以及包含此元件的病毒基因组转录盒及载体。The present disclosure relates to the field of viral vectors, in particular, to the field of retroviruses, especially lentiviral vectors; more specifically, to the initiation of transcription in viral genome transcription cassettes that carry target nucleic acid fragments for preparing retroviruses, especially lentiviral vectors. Elements, and viral genome transcription cassettes and vectors containing this element.
背景技术Background technique
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。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. 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. 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. 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.
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。γ-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. At present, 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 %. This is because lentivirus can transduce non-dividing cells, which distinguishes it from other viral vectors (including gamma-retroviral vectors). In addition, lentiviruses have a more favorable gene insertion site lineage compared to retroviruses. The most attractive features of 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.
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)。The prototype lentiviral vector system was developed based on the HIV-1 virus, which is a human pathogen virus that has been fully studied. In addition to HIV-1, other lentiviruses have also been developed as gene delivery vectors (TV), but most of them have not reached the stage of clinical research, such as 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). A vector based solely on Equine infectious anemia virus (EIAV) has been developed to the clinical use stage (ProSavin) for the treatment of Parkinson's.
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(SV40polyA)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。Mainly out of consideration of the safety issues caused by the pathogenicity of HIV-1 in humans, three generations of lentiviral vector systems have been developed. 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, and its 5'-end LTR is replaced by the cytomegalovirus early promoter, and the 3'-LTR is replaced by the simian virus-40 polyadenylic acid (SV40polyA) sequence, and the HIV-1 Envelope gene env. 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. In addition, 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. At the same time, deleting the U3 region also greatly reduces the carcinogenicity of the vector gene inserted into the host cell. 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.
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。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 (Mokola virus), etc.) envelope protein, filamentous virus (Filoviruses, such as Ebola virus, Zaire virus, etc.) envelope protein, trachoma virus (Arenaviruses, such as lymphocytes) Choroidal meningitis virus (Lymphocytic choriomeningitis virus, etc.) envelope protein, baculovirus (Baculovirus) envelope protein, alphavirus (Alphaviruses, such as Chikungunya virus, Ross River virus), Semliki Forest virus, Sindbis virus, Venezuelan equi encephalitis virus, Western equi encephalitis virus, etc.) envelope protein, Orthomyxoviruses, such as influenza virus (Influenza virus), avian plague virus (Fowl Plag) ue Virus, etc.) envelope proteins, herpes viruses (Vesiculoviruses, such as Vesicular stomatitis virus, Chandipura virus and Piry virus, etc.) envelope proteins are currently large Most lentiviral vectors are prepared using vesicular stomatitis virus envelope glycoprotein (VSV-G). This is because this glycoprotein allows the lentiviral vector to have a wide range of transduction spectrum and downstream Better stability during processing.
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、Hela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。At present, most 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. In addition, compared with HEK293 cells, 293T cells have a faster cell growth rate and higher transfection efficiency. The above 293T cell characteristics make it have a higher efficiency of virus vector preparation. In addition, according to literature reports, the production/packaging cells that can be used for viral vectors also include mammalian cells HepG2 cells, Hela cells, CHO cells, BHK cells, COS cells, NIH/3T3 cells, Vero cells, HT1080 cells, Te671 cells, CEM Cells, NSO cells and PerC6 cells.
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核酸片段的构建体比如质粒，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核酸片段形成稳定 生产细胞系(producer cell line)以进行持续生产来实现(稳定生产)。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, ψ 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.). This can be achieved by transiently transfecting a construct containing the aforementioned nucleic acid fragment, such as a plasmid, into the aforementioned host cell, followed by 24-72 hours of viral vector production and harvesting (transient production), or by transfecting the aforementioned host cell genome Stable integration of the above-mentioned nucleic acid fragments to form a stable producer cell line for continuous production (stable production).
当前使用的大多数逆转录病毒载体均来自莫洛尼氏鼠白血病病毒(Mo-MLV)，此载体系统多年来已实现了许多设计改进。快速高效制备逆转录病毒载体的方法是通过瞬时转染基于293的包装细胞系，例如Phoenix-ECO和Phoenix-AMPHO(Dr.Gary P.Nolan,Stanford University)或其它商业可用的包装细胞系如Plat-A，Plat-E，和Plat-GP(Cell Biolabs)；AmpphoPak-293；EcoPak2-293；和RetroPack PT67(Clonteck)。为了将逆转录病毒载体与其他包膜蛋白(如水泡性口炎病毒糖蛋白(VSV-G))进行假型化，可以在仅表达Mo-MLV gag和pol基因的包装细胞系(例如Plat-GP，Cell Biolabs；GP2-293，Clontech)中同时转染转移载体质粒和表达目的包膜蛋白的质粒；或者通过将表达Mo-MLV gag和pol基因的质粒(例如pUMVC-质粒，Addgene，#8449)、表达目的包膜蛋白的质粒和转移载体质粒同时瞬时转染293细胞。Most of the retroviral vectors currently in use are derived from Moloney Murine Leukemia Virus (Mo-MLV), and this vector system has achieved many design improvements over the years. The rapid and efficient method of preparing retroviral vectors is by transiently transfecting 293-based packaging cell lines, such as Phoenix-ECO and Phoenix-AMPHO (Dr. Gary P. Nolan, Stanford University) or other commercially available packaging cell lines such as Plat. -A, Plat-E, and Plat-GP (Cell Biolabs); AmpphoPak-293; EcoPak2-293; and RetroPack PT67 (Clonteck). In order to pseudotype the retroviral vector with other envelope proteins (such as vesicular stomatitis virus glycoprotein (VSV-G)), it can be used in packaging cell lines that only express Mo-MLV gag and pol genes (such as Plat- GP, Cell Biolabs; GP2-293, Clontech) were simultaneously transfected with a transfer vector plasmid and a plasmid expressing the target envelope protein; or by combining a plasmid expressing Mo-MLV gag and pol genes (such as pUMVC-plasmid, Addgene, #8449 ), the plasmid expressing the target envelope protein and the transfer vector plasmid are transiently transfected into 293 cells at the same time.
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 PCTCN2020115521-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。 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. Other methods can also be used, such as calcium phosphate precipitation, cationic liposome complexation and non-liposomal transfection reagents, such as Lipofectamine and
Figure PCTCN2020115521-appb-000001
However, these methods can only be used for small-scale production or only for research purposes because they are either difficult to scale up or too expensive. Alternatively, viral infections have also been developed and validated for retroviral/lentiviral vector production, which uses baculovirus or adenovirus to introduce lentiviral gene constructs. However, this method requires additional downstream separation of retroviral/lentiviral vectors and baculovirus or adenovirus to meet clinical-grade virus production standards, and compared to plasmid DNA transfection methods, baculoviruses are used. Or adenovirus to prepare retroviral/lentiviral vector is complicated and the final transfection titer is not high. Alternatively, 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.
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。The construction of a stable retrovirus/lentivirus production cell line relies on the integration of viral gene constructs into the cell genome to allow constitutive expression or regulated expression. Usually, 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. , So as to establish a packaging cell line. Afterwards, 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. If a non-SIN (self-inactivating) vector that can be replicated after reverse transcription is used, this can be achieved directly by viral infection; otherwise, it needs to be transfected with plasmid chemical followed by resistance screening and clone selection to obtain the above-mentioned packaging cells A production cell line that stably integrates the viral genome transcription cassette carrying the target nucleic acid fragment into the genome. 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. This method has been shown to allow the establishment of high-titer human retroviral vector production cell lines (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.,etal.(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.). In addition, similar principles can be used to develop viral vector packaging cell lines or production cell line platforms for rapid replacement of target nucleic acid fragments and/or outer envelope proteins. The development of stable retroviral/lentiviral vector production cell lines is a time-consuming and complex system engineering, which requires a year or more to fully develop and characterize the cell line platform, and due to the complexity of the work, Many of the published work ultimately failed to meet industry needs due to issues such as toxin production titer, cell line stability, and culture adaptation. However, as compensation, once a stable virus production cell line is successfully developed and developed, in clinical and industrial application fields, stable production cell lines are compared to transient transfection production in terms of process reliability, process amplification capability, production cost, and virus product safety. The process has irreplaceable advantages. First, 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. When expanding the scale of production, 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.
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。据估计基于现有生产技术平台，每位患者需要约10-100L培养体积生产的病毒载体，因此，目前的逆转录病毒和慢病毒载体生产系统的表现是远低于治疗需求的，任何提高现有逆转录病毒和慢病毒载体生产系统的生产能力都是极富价值的。 Present before purification cell stock can be obtained in the production of retroviral / lentiviral vector titers of 10 6 to 10 7 infectious particles / mL medium. However, the average amount of vector needed to treat a patient in a clinical trial is at the level of 10 10 infectious particles per patient. In addition, 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. It is estimated that based on the existing production technology platform, each patient needs about 10-100L of viral vectors produced in a culture volume. Therefore, the performance of the current retroviral and lentiviral vector production systems is far below the demand for treatment. The production capacity of retroviral and lentiviral vector production systems is extremely valuable.
因此，本领域中目前迫切地需要提供一种提高逆转录病毒和慢病毒载体生产能力的技术。Therefore, there is an urgent need in the art to provide a technology for improving the production capacity of retrovirus and lentiviral vectors.
技术问题technical problem
本公开通过构建使用Tet-On诱导表达系统Tet响应元件(TRE)调控携带目的核酸片段的逆转录病毒/慢病毒基因组转录盒的转录从而显著提高了逆转录病毒/慢病毒载体的生产滴度。The present disclosure significantly increases the production titer of the retrovirus/lentiviral vector by constructing a Tet-responsive element (TRE) that uses the Tet-On inducible expression system to regulate the transcription of a retroviral/lentiviral genome transcription cassette carrying a target nucleic acid fragment.
技术解决方案Technical solutions
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和LTR构建的嵌合启动子)，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(比如将上述转录盒构建到转移载体质粒中或病毒载体中)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。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 fragments (such as primers or probes) RNA sequence; can be any nucleic acid sequence from nature or man-made nucleic acid sequence; it can also be a combination of the above one or more nucleic acid sequences; the target nucleic acid fragment can also include nucleic acid sequences that regulate gene expression such as promoters, enhancers, and isolation Sequences such as nucleus, polyadenylic acid signal and so on. 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 ψ 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. When preparing retrovirus/lentiviral vectors, 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 self-LTR sequence or a chimeric promoter constructed by other heterologous promoters and LTR), a DNA sequence corresponding to the viral RNA genome, and usually a polyadenylation signal sequence that regulates the termination of transcription. When preparing a retrovirus/lentiviral vector, a construct carrying a viral genome transcription cassette carrying a nucleic acid fragment of interest (for example, constructing the above transcription cassette into a transfer vector plasmid or a viral vector) is delivered to the host cell, Through the transcriptional molecular mechanism of host cells, it is transcribed into corresponding viral genome RNA fragments that can be packaged into viral vectors and carry target nucleic acid fragments.
携带目的核酸片段的逆转录病毒基因组转录盒中需提供转录、包装和转导所需的顺式 作用元件，主要包括：(1)长末端重复(long terminal repeats，LTR)片段：LTR的主要功能是调节病毒基因组从RNA逆转录至DNA、DNA前病毒(provirus)在宿主细胞基因组整合以及整合后转录病毒mRNA片段。LTR由U3(unique-3’)、R(repeat)和U5(unique-5’)三个功能域按照U3-R-U5的结构组成，其中U3功能域主要提供转录启动子和增强子功能；R功能域参与反转录过程，并且编码病毒RNA基因组的5'加帽序列(5'cap)和聚腺苷酸化(polyA)信号；U5功能域是逆转录的起始位点。目前常用的SIN载体删除了3’LTR中的U3功能域，并通过外源启动子和5’LTR中的R-U5功能域组成的嵌合启动子调控整个携带目的核酸片段的病毒RNA基因组转录盒的转录。(2)引物结合位点(primer binding site,PBS):位于5'LTR下游，紧靠近U5功能域，与宿主细胞内的特定转运RNA(tRNA)结合，并以此tRNA为引物启动反转录。(3)ψ包装信号(ψpackaging signal)：位于引物结合位点和gag开放阅读框之间，是病毒RNA基因组包装进病毒颗粒内的必要元件。(4)多嘌呤束(polypurine tract，PPT):位于3’LTR上游，紧靠近U3功能域，是反转录过程中正链DNA合成的引物。对于慢病毒基因组转录盒，还需提供下列必要顺式作用元件：(1)中央多嘌呤束/中央终止序列(cPPT/CTS)：位于慢病毒基因组的第二条多嘌呤束，对于前病毒转运进入宿主细胞核至关重要，能显著提高前病毒复合体整合进宿主细胞基因组的效率。(2)Rev响应元件(Rev response element,RRE):与慢病毒编码的调节蛋白Rev结合，该蛋白对于转录后的未剪接和不完全剪接的病毒mRNA从细胞核到细胞质的转运至关重要。慢病毒基因组转录盒中包含RRE序列能显著提高慢病毒载体的包装效率。除此之外，在逆转录病毒/慢病毒载体的基因组转录盒中的目的核酸片段3’端连接转录后调控元件(posttranscriptional regulatory elements)可以显著提高目的核酸片段中转基因的表达水平，例如连接土拨鼠肝炎病毒的转录后调控元件(Woodchuck Hepatitis Virus(WHV)Posttranscriptional Regulatory Element，WPRE)或梅森-辉瑞猴病毒(Mason-Pfizer monkey virus)的组成型转运元件(Constitutive Transport Element,CTE)。The retroviral genome transcription cassette carrying the target nucleic acid fragment needs to provide the cis-acting elements required for transcription, packaging and transduction, mainly including: (1) Long terminal repeats (LTR) fragments: the main function of LTR It regulates the reverse transcription of viral genome from RNA to DNA, DNA provirus integration in the host cell genome and transcription of viral mRNA fragments after integration. LTR is composed of three functional domains of U3 (unique-3'), R (repeat) and U5 (unique-5') according to the structure of U3-R-U5, and the U3 functional domain mainly provides transcription promoter and enhancer functions; The R functional domain participates in the reverse transcription process and encodes the 5'capping sequence (5'cap) and polyadenylation (polyA) signal of the viral RNA genome; the U5 functional domain is the start site of reverse transcription. The currently commonly used SIN vector deletes the U3 functional domain in the 3'LTR, and regulates the transcription of the entire viral RNA genome carrying the target nucleic acid fragment through a chimeric promoter composed of an exogenous promoter and the R-U5 functional domain in the 5'LTR Transcription of the box. (2) Primer binding site (PBS): Located downstream of the 5'LTR, close to the U5 functional domain, it binds to the specific transfer RNA (tRNA) in the host cell, and uses the tRNA as a primer to initiate reverse transcription . (3) ψ packaging signal: Located between the primer binding site and the gag open reading frame, it is a necessary element for the packaging of viral RNA genome into viral particles. (4) Polypurine tract (PPT): Located upstream of 3'LTR, close to the U3 functional domain, it is a primer for the synthesis of positive strand DNA during reverse transcription. For the lentiviral genome transcription cassette, the following necessary cis-acting elements must also be provided: (1) Central polypurine tract/central termination sequence (cPPT/CTS): the second polypurine tract located in the lentiviral genome, for proviral transport Entering the nucleus of the host cell is critical and can significantly improve the efficiency of integration of the proviral complex into the host cell genome. (2) Rev response element (RRE): binds to the regulatory protein Rev encoded by the lentivirus, which is essential for the transport of unspliced and incompletely spliced viral mRNA from the nucleus to the cytoplasm after transcription. The inclusion of the RRE sequence in the lentiviral genome transcription cassette can significantly improve the packaging efficiency of the lentiviral vector. In addition, the 3'end of the target nucleic acid fragment in the genome transcription cassette of the retroviral/lentiviral vector is connected with posttranscriptional regulatory elements (posttranscriptional regulatory elements) can significantly increase the expression level of the transgene in the target nucleic acid fragment. The post-transcriptional regulatory element (Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element, WPRE) or the Constitutive Transport Element (CTE) of Mason-Pfizer monkey virus (Mason-Pfizer monkey virus).
术语“载体”是一种包含核酸分子，通常被用作将外来遗传物质，如上述目的核酸片段，人工携带到另一个细胞中的媒介物，并且在其中复制和/或表达。在功能上，所有载体一般都可以用于克隆并携带外来目的核酸片段，同时也有专门设计的用于核酸片段转录和蛋白质表达的表达载体。质粒，病毒载体，粘粒(cosmids)和人工染色体是载体的四种主要类型。其中，最常用的载体是质粒。所有工程质粒载体都包含在细菌中复制的复制起点，用于插入目的核酸片段的多克隆位点和用于选择阳性菌株的标记基因。病毒载体是另一种常用载体，通常用来将遗传物质，如目的核酸片段输送到细胞中。此过程可以在生物体内(体内)或细胞培养物中(体外)进行。基于病毒自身进化出的多种分子机制，如对遗传 物质的保护、基于受体对宿主细胞的选择、遗传物质向宿主细胞内的递送、在宿主细胞内的复制和/或表达、对宿主细胞生长、代谢、繁殖复制和防御机制的调整、以及在高等动物体内对免疫系统的抑制和/或逃逸，可以有效地将目的核酸片段转移到感染的靶细胞内。除了被用于分子生物学研究中，病毒载体还常被用于基因治疗、细胞治疗、免疫治疗和疫苗开发。在本申请中，用于包装逆转录病毒/慢病毒的基因片段和携带目的核酸片段的病毒基因组转录盒可以通过构建上述各种载体并转入宿主细胞进行病毒载体生产。The term "vector" refers to a nucleic acid molecule, which is usually used as a vehicle for artificially carrying foreign genetic material, such as the above-mentioned nucleic acid fragment of interest, into another cell, where it is replicated and/or expressed. Functionally, all vectors can generally be used to clone and carry foreign nucleic acid fragments of interest, and there are also specially designed expression vectors for nucleic acid fragment transcription and protein expression. Plasmids, viral vectors, cosmids and artificial chromosomes are the four main types of vectors. Among them, the most commonly used vector is a plasmid. All engineered plasmid vectors contain an origin of replication in bacteria, a multiple cloning site for inserting target nucleic acid fragments, and a marker gene for selecting positive strains. Viral vectors are another commonly used vector, usually used to deliver genetic material, such as nucleic acid fragments of interest, into cells. This process can be performed in vivo (in vivo) or in cell culture (in vitro). A variety of molecular mechanisms based on the evolution of the virus itself, such as the protection of genetic material, the selection of host cells based on the receptor, the delivery of genetic material into the host cell, the replication and/or expression in the host cell, the protection of the host cell The adjustment of growth, metabolism, reproduction and defense mechanisms, as well as the suppression and/or escape of the immune system in higher animals, can effectively transfer target nucleic acid fragments to infected target cells. In addition to being used in molecular biology research, viral vectors are often used in gene therapy, cell therapy, immunotherapy, and vaccine development. In this application, the gene fragments used for packaging retrovirus/lentivirus and the viral genome transcription cassette carrying the target nucleic acid fragment can be produced by constructing the above-mentioned various vectors and transferring them into host cells for viral vector production.
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携带目的核酸片段的病毒基因组转录盒的转录。在本公开中，可用的诱导表达系统的实例包括，但不限于，Tet-off系统(参见，例如，Gossen,M.和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))、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))。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:11中13-756bp)和TRE adv)以及第三代Tet-On诱导表达系统(Tet-On 3G，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:10中13-756bp)和TRE 3G)。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。 In the present disclosure, an inducible expression system is used to control the transcription of a viral genome transcription cassette carrying a target nucleic acid fragment into a host cell. In the present disclosure, examples of usable inducible expression systems include, but are not limited to, the Tet-off system (see, for example, Gossen, M. and H. Bujard (1992). "Tight control of gene expression in mammalian cells by tetracycline -responsive promoters."Proc Natl Acad Sci USA 89(12):5547-5551; Yu,H.,et al.(1996)."Inducible human immunodeficiency virus type 1 packaging 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 (the above documents are incorporated herein by reference)) , Tet-On inducible expression system (see, for example, WO0075347A2, WO2007058527A2 (the above documents are incorporated herein by reference)). 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. Currently commonly used 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: 13-756bp in SEQ ID NO: 11) and TRE adv ) and the third generation Tet-On inducible expression system (Tet-On 3G, Clontech, in which the transactivator rtTA and the response element TRE are respectively referred to as rtTA 3G hereinafter (Encoding nucleic acid sequence: 13-756bp in SEQ ID NO: 10) and TRE 3G ). 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 .
本公开涉及在逆转录病毒/慢病毒载体生产过程中，基于四环素依赖的核酸序列(Tet响应元件，TRE)调控携带目的核酸片段的病毒基因组转录盒转录。具体地，所述Tet响应元件TRE包含至少2个拷贝的结合四环素依赖的反式激活物(reverse tetracycline controlled transactivator，rtTA)的TetO-操作子序列(Tet operator，TetO)、1个拷贝的包含TATA盒序列的最小启动子序列；优选地，所述TRE序列如SEQ ID NO:17和SEQ ID NO:18所示。在本公开的一个方面，调控携带目的核酸片段的病毒基因组转录盒转录的嵌合启动子包含 上述Tet响应元件TRE和逆转录病毒/慢病毒的LTR序列中的R-U5功能域；优选地，R-U5功能域连接在Tet响应元件中的TATA盒序列下游并与其间隔20-24个碱基对，更优选地间隔24个碱基对。在本公开的一个方面，所述R-U5功能域是HIV-1慢病毒的R-U5功能域，优选地，所述Tet响应元件和HIV-1慢病毒的R-U5功能域组成的嵌合响应元件为TRE1-RU5(如SEQ ID NO:19所示)和TRE2-RU5(如SEQ ID NO:20所示)。The present disclosure relates to the regulation of transcription cassettes of viral genomes carrying target nucleic acid fragments based on tetracycline-dependent nucleic acid sequences (Tet response elements, TRE) during the production process of retroviral/lentiviral vectors. Specifically, the Tet response element TRE includes at least 2 copies of a TetO-operator sequence (TetO) that binds to a tetracycline-dependent transactivator (reverse tetracycline controlled transactivator, rtTA), and 1 copy of a TATA The minimal promoter sequence of the box sequence; preferably, the TRE sequence is shown in SEQ ID NO: 17 and SEQ ID NO: 18. In one aspect of the present disclosure, the chimeric promoter that regulates the transcription of the viral genome transcription cassette carrying the nucleic acid fragment of interest includes the above-mentioned Tet response element TRE and the R-U5 functional domain in the LTR sequence of the retrovirus/lentivirus; preferably, The R-U5 functional domain is connected downstream of the TATA box sequence in the Tet response element and is separated from it by 20-24 base pairs, more preferably 24 base pairs. In one aspect of the present disclosure, the R-U5 functional domain is the R-U5 functional domain of the HIV-1 lentivirus. Preferably, the Tet response element and the R-U5 functional domain of the HIV-1 lentivirus are embedded The combined response elements are TRE1-RU5 (as shown in SEQ ID NO: 19) and TRE2-RU5 (as shown in SEQ ID NO: 20).
在本公开中，携带目的核酸片段的γ-逆转录病毒基因组转录盒可以来自使用5’LTR做为启动子的MSCV(小鼠干细胞病毒，Murine Stem Cell Virus)逆转录病毒表达系统的转移载体质粒，例如pMSCV-IRES-mCherry FP(Addgene，#52114)、pMSCV-IRES-GFP(Addgene，#20672)；来在使用5’LTRz做为启动子的MoMLV(莫洛尼鼠白血病病毒，Moloney Murine Leukemia Virus)逆转录表达系统的转移载体质粒，例如pBABE-hygro-hTERT(Addgene，#1773)、pBABE-puro(Addgene，#1764)；以及以上两种逆转录表达系统的基于CMV-R-U5嵌合启动子的并且删除U3功能域的SIN转移载体质粒，例如pMKO.1GFP(Addgene，#10676)，TtRMPVIR(Addgene，#27995)，pRetroX GFP T2A Cre(Addgene，#63704)，pRXTN(Addgene，#47916)。上述逆转录病毒转移载体质粒中的病毒基因组转录盒在原理上都适用本公开所描述的使用Tet响应元件调控病毒基因组转录盒转录的方法。In the present disclosure, the γ-retroviral genome transcription cassette carrying the target nucleic acid fragment can be derived from the transfer vector plasmid of the MSCV (Murine Stem Cell Virus) retroviral expression system using 5'LTR as the promoter For example, pMSCV-IRES-mCherry FP (Addgene, #52114), pMSCV-IRES-GFP (Addgene, #20672); to use 5'LTRz as the promoter of MoMLV (Moloney Murine Leukemia) Virus) reverse transcription expression system transfer vector plasmid, such as pBABE-hygro-hTERT (Addgene, #1773), pBABE-puro (Addgene, #1764); and the above two reverse transcription expression systems based on CMV-R-U5 embedded The SIN transfer vector plasmid that combines the promoter and deletes the U3 domain, such as pMKO.1GFP (Addgene, #10676), TtRMPVIR (Addgene, #27995), pRetroX GFP T2A Cre (Addgene, #63704), pRXTN (Addgene, # 47916). The viral genome transcription cassettes in the above-mentioned retroviral transfer vector plasmids are in principle applicable to the method described in the present disclosure using Tet response elements to regulate transcription of viral genome transcription cassettes.
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通过被Tet响应元件调控的携带目的核酸片段的病毒 基因组转录盒制备病毒载体的方法。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。In the present disclosure, 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 Ψ packaging signal, RRE, cPPT and gag partial sequences, etc. in virus packaging and transduction The nucleic acid sequence that plays a key role in the process. 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. In the third-generation lentiviral genome transcription cassette, pSLIK-Hygro, pHIV-EGFP, and pSico vectors use the CMV promoter to transcribe lentiviral genomic RNA, while pRRLSIN.cPPT.PGK-GFP.WPRE, Tet-pLKO-puro, pLenti -Puro uses the RSV promoter to transcribe lentiviral genomic RNA. Compared with other third-generation lentiviral transfer vector plasmids, Tet-pLKO-puro and pLenti-puro do not contain WPRE sequence in the lentiviral genome transcription cassette. The lentiviral genome transcription cassettes in the above-mentioned lentiviral transfer vector plasmids are in principle applicable to the method of preparing viral vectors through the viral genome transcription cassettes carrying the target nucleic acid fragments regulated by Tet response elements described in the present disclosure. In one aspect of 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.
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。In the present disclosure, 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 virus, Feline leukemia virus (Feline leukemia virus), both sexes Amphotropic retrovirus, Ecotropic retrovirus, Baboon ape leukemia virus, and all other Retroviridae viruses.
逆转录病毒/慢病毒载体的制备可以通过在宿主细胞内转入gag基因、pol基因、rev基因(用于慢病毒载体)、包膜糖蛋白基因，以及本公开所述被Tet响应元件调控转录的携带目的核酸片段的病毒基因组转录盒(同时需要转入调控Tet响应元件的反式激活物rtTA)来实现。上述逆转录病毒/慢病毒载体生产方法可以通过“瞬时生产”来实现，其表现为将含有本公开所述被Tet响应元件调控转录的携带目的核酸片段的病毒基因组转录盒(以下简称病毒基因组转录盒)的载体，如质粒或病毒载体，转入宿主细胞内，并在没有整合进宿主细胞基因组的情况下制备病毒载体。在本公开的一个方面，将包含gag基因、pol基因、rev基因(用于慢病毒载体)、包膜糖蛋白基因、反式激活物rtTA编码序列以及病毒基因组转录盒的载体构造物(如质粒或病毒载体)全部通过瞬时转染的方法转入宿主细胞制备逆转录病毒/慢病毒载体。在本公开的一个方面，先将反式激活物rtTA编码序列稳定整合在宿主细胞的基因组中，使得宿主细胞稳定表达rtTA蛋白。然后基于此基因改造后的宿主细胞，通过将包含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病毒基因组转录盒的载体构造物(如质粒或病毒载体)全部通过瞬时转染的方法转入宿主细胞制备逆转录病毒/慢病毒载体。在本公开的一个方面，先将反式激活物rtTA编码序列稳定整合在宿主细胞的基因组中，同时将gag基因、pol基因、rev基因(用于慢病毒载体)、包膜糖蛋白基因的一种，多种或全部稳定整合在宿主细胞的基因组中构建包装细胞系，然后通过将未稳定整合在包装细胞中的gag基因、pol基因、rev基因(用于慢病毒载体)和包膜糖蛋白基因以载体构造物(如质粒或病毒载体)的形式和病毒基因组转录盒的载体构造物(如质 粒或病毒载体)同时通过瞬时转染的方法转入上述包装细胞制备逆转录病毒/慢病毒载体。优选地，先将反式激活物rtTA编码序列、gag基因、pol基因、rev基因(用于慢病毒载体)、包膜糖蛋白基因全部稳定整合在宿主细胞的基因组中构建包装细胞。然后将病毒基因组转录盒通过瞬时转染的方法转入上述包装细胞制备逆转录病毒/慢病毒载体。在以上瞬时生产方法中，需加入四环素或其衍生物诱导制备逆转录病毒/慢病毒载体。上述逆转录病毒/慢病毒载体生产方法也可以通过“稳定生产”来实现，其表现为将含有本公开所述被Tet响应元件调控转录的携带目的核酸片段的病毒基因组转录盒(以下简称病毒基因组转录盒)连同包装基因如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反式激活物rtTA编码序列全部稳定整合在宿主细胞的基因组中构建生产细胞系，并通过添加四环素或其衍生物诱导制备逆转录病毒/慢病毒载体。在本公开的一个方面，上述构建逆转录病毒/慢病毒载体的包装细胞和生产细胞可以通过转座子系统如睡美人(Sleeping Beauty，SB)转座子系统和/或PiggyBac(PB)转座子系统实现。Retroviral/lentiviral vectors can be prepared by introducing gag genes, pol genes, rev genes (for lentiviral vectors), envelope glycoprotein genes, and transcription regulation by Tet response elements in the host cell. The viral genome transcription cassette carrying the target nucleic acid fragment (at the same time needs to be transferred to the transactivator rtTA that regulates the Tet response element). The above-mentioned retroviral/lentiviral vector production method can be realized by "transient production", which is expressed as a viral genome transcription cassette (hereinafter referred to as viral genome transcription) containing a target nucleic acid fragment regulated by Tet response element transcription as described in the present disclosure. Box) vectors, such as plasmids or viral vectors, are transformed into host cells, and viral vectors are prepared without integration into the host cell genome. In one aspect of this disclosure, a vector construct (such as a plasmid Or viral vectors) are all transformed into host cells by transient transfection to prepare retroviral/lentiviral vectors. In one aspect of the present disclosure, the transactivator rtTA coding sequence is first stably integrated into the genome of the host cell, so that the host cell stably expresses the rtTA protein. Then based on this genetically modified host cell, a vector construct (such as a plasmid or a viral vector) containing the gag gene, pol gene, rev gene (for lentiviral vectors), envelope glycoprotein gene, and viral genome transcription cassette All were transformed into host cells by transient transfection to prepare retroviral/lentiviral vectors. In one aspect of the present disclosure, the transactivator rtTA coding sequence is first stably integrated into the genome of the host cell, and at the same time one of the gag gene, pol gene, rev gene (for lentiviral vectors), and envelope glycoprotein gene is integrated. Species, multiple or all of them are stably integrated into the genome of the host cell to construct a packaging cell line, and then the gag gene, pol gene, rev gene (for lentiviral vector) and envelope glycoprotein that are not stably integrated in the packaging cell Genes in the form of vector constructs (such as plasmids or viral vectors) and the vector constructs of viral genome transcription cassettes (such as plasmids or viral vectors) are simultaneously transferred into the above-mentioned packaging cells by transient transfection to prepare retrovirus/lentiviral vectors . Preferably, the transactivator rtTA coding sequence, gag gene, pol gene, rev gene (for lentiviral vector), and envelope glycoprotein gene are all stably integrated into the genome of the host cell to construct a packaging cell. Then, the viral genome transcription cassette is transformed into the above-mentioned packaging cell by transient transfection method to prepare a retrovirus/lentiviral vector. In the above transient production method, tetracycline or its derivatives need to be added to induce the preparation of retroviral/lentiviral vectors. The above-mentioned retroviral/lentiviral vector production method can also be achieved by "stable production", which is represented by the expression of a viral genome transcription cassette (hereinafter referred to as viral genome) containing a target nucleic acid fragment regulated by the Tet response element in the present disclosure. Transcription cassette) together with packaging genes such as gag gene, pol gene, rev gene (for lentiviral vectors), envelope glycoprotein gene and transactivator rtTA coding sequence are all stably integrated into the genome of the host cell to construct a production cell line, And by adding tetracycline or its derivatives to induce preparation of retroviral/lentiviral vectors. In one aspect of the present disclosure, the above-mentioned packaging cells and production cells for constructing retroviral/lentiviral vectors can be transposed by a transposon system such as Sleeping Beauty (SB) transposon system and/or PiggyBac (PB) Subsystem implementation.
在本公开中，可用于制备逆转录病毒/慢病毒载体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、Hela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，宿主细胞可以被贴壁培养或悬浮培养。在一个方面，宿主细胞可以在有血清或无血清添加的条件下培养。In the present disclosure, host cells that can be used to prepare retroviral/lentiviral vectors are mammalian cells. Examples of host cells suitable for use in the present disclosure are 293T cells, HepG2 cells, Hela 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. In one aspect, the host cell is a HEK293 cell or a cell derived from a HEK293 cell. In one aspect, the host cell is a 293T cell. In one aspect, host cells can be cultured adherently or in suspension. In one aspect, the host cell can be cultured with or without addition of serum.
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。In the present disclosure, 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 10-6M; preferably, its binding constant Ka reaches or is stronger than 10-9M. The tetracycline derivative may be selected from, for example, doxycycline (Dox), anhydrotetracycline (Atc), chlortetracycline, oxytetracycline, and deoxytetracycline.
在一个方面，本公开提供以下各项：In one aspect, the present disclosure provides the following:
第1项.核酸序列，其包含Tet-On系统的响应元件TRE和逆转录病毒长末端重复序列(LTR)中的R-U5功能域。Item 1. Nucleic acid sequence, which contains the response element TRE of the Tet-On system and the R-U5 functional domain in the retroviral long terminal repeat (LTR).
第2项.第1项的核酸序列，其中所述R-U5功能域在TRE的TATA盒下游并与其间隔15-30bp。Item 2. The nucleic acid sequence of item 1, wherein the R-U5 functional domain is downstream of the TATA box of the TRE and separated from it by 15-30 bp.
第3项.第2项的核酸序列，其中所述R-U5功能域在TRE的TATA盒下游并与其间隔24bp。Item 3. The nucleic acid sequence of item 2, wherein the R-U5 functional domain is downstream of the TATA box of the TRE and separated from it by 24 bp.
第4项.第1项的核酸序列，其中TRE的序列如SEQ ID NO:17或SEQ ID NO:18所 示。Item 4. The nucleic acid sequence of item 1, wherein the sequence of TRE is shown in SEQ ID NO: 17 or SEQ ID NO: 18.
第5项.第1项的核酸序列，其序列如SEQ ID NO:19或SEQ ID NO:20所示。Item 5. The nucleic acid sequence of item 1, whose sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 20.
第6项.逆转录病毒基因组转录盒，其包含用于控制所述转录盒的转录的Tet-On系统的响应元件TRE或第1-5项中任一项的核酸序列，位于Tet-On系统的响应元件TRE或第1-5项中任一项的核酸序列下游的用于逆转录病毒包装的顺式作用元件以及用于插入目的核酸片段的多克隆位点。Item 6. A retroviral genome transcription cassette, which contains the Tet-On system response element TRE or the nucleic acid sequence of any one of items 1-5 for controlling the transcription of the transcription cassette, located in the Tet-On system The response element TRE or the cis-acting element used for retrovirus packaging downstream of the nucleic acid sequence of any one of items 1-5 and a multiple cloning site for inserting the target nucleic acid fragment.
第7项.第6项的逆转录病毒基因组转录盒，其中所述顺式作用元件包括长末端重复序列(LTR)、引物结合位点(PBS)和病毒包装信号(phi信号)。Item 7. The retroviral genome transcription cassette of Item 6, wherein the cis-acting element includes a long terminal repeat (LTR), a primer binding site (PBS) and a viral packaging signal (phi signal).
第8项.第6项的逆转录病毒基因组转录盒，其中所述逆转录病毒是慢病毒。Item 8. The retroviral genome transcription cassette of item 6, wherein the retrovirus is a lentivirus.
第9项.第8项的逆转录病毒基因组转录盒，其中所述顺式作用元件包括长末端重复序列(LTR)、引物结合位点(PBS)、病毒包装信号(phi信号)、中心多嘌呤束(cPPT)和rev蛋白应答元件(RRE)。Item 9. The retroviral genome transcription cassette of Item 8, wherein the cis-acting element includes a long terminal repeat (LTR), a primer binding site (PBS), a viral packaging signal (phi signal), and a central polypurine Bundle (cPPT) and rev protein response element (RRE).
第10项.第8项的逆转录病毒基因组转录盒，其中所述顺式作用元件还包括土拨鼠肝炎病毒转录后调控序列(WPRE)。Item 10. The retroviral genome transcription cassette of Item 8, wherein the cis-acting element further includes a woodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE).
第11项.第7项或第9项的逆转录病毒基因组转录盒，其中所述长末端重复序列是能自我复制的野生型U3-R-U5序列或是删除了U3序列的自我抑制SIN序列。Item 11. The retroviral genome transcription cassette of item 7 or 9, wherein the long terminal repeat sequence is a wild-type U3-R-U5 sequence capable of self-replication or a self-suppressive SIN sequence with U3 sequence deleted .
第12项.逆转录病毒基因组转录盒，其通过在第6-11项中任一项的逆转录病毒基因组转录盒的多克隆位点中插入目的核酸片段获得。Item 12. Retroviral genome transcription cassette, which is obtained by inserting a nucleic acid fragment of interest into the multiple cloning site of the retroviral genome transcription cassette of any one of items 6-11.
第13项.载体，所述载体包含第1-5项中任一项的核酸序列或第6-12项中任一项的逆转录病毒基因组转录盒。Item 13. A vector comprising the nucleic acid sequence of any one of items 1-5 or the retroviral genome transcription cassette of any one of items 6-12.
第14项.第13项所述的载体，其是质粒载体或病毒载体。Item 14. The vector of item 13, which is a plasmid vector or a viral vector.
第15项.宿主细胞，所述宿主细胞包含第1-5项中任一项的核酸序列、第6-12项中任一项的逆转录病毒基因组转录盒或第13项或第14项的载体。Item 15. A host cell comprising the nucleic acid sequence of any one of items 1-5, the retroviral genome transcription cassette of any one of items 6-12, or the 13th or 14th item Carrier.
第16项.第1-5项中任一项的核酸序列、第6-12项中任一项的逆转录病毒基因组转录盒、第13项或第14项的载体、或第15项的宿主细胞用于生产携带目的核酸片段的逆转录病毒载体的用途。Item 16. The nucleic acid sequence of any one of items 1-5, the retroviral genome transcription cassette of any one of items 6-12, the vector of item 13 or 14, or the host of item 15 Cells are used to produce retroviral vectors carrying target nucleic acid fragments.
第17项.第16项的用途，其中第1-5项中任一项的核酸序列、第6-12项中任一项的逆转录病毒基因组转录盒、第13项或第14项的载体、或第15项的宿主细胞被用于所述携带目的核酸片段的逆转录病毒载体的瞬时生产或稳定生产。Item 17. The use of item 16, wherein the nucleic acid sequence of any one of items 1-5, the retroviral genome transcription cassette of any one of items 6-12, and the vector of item 13 or 14 , Or the host cell of item 15 is used for transient or stable production of the retroviral vector carrying the nucleic acid fragment of interest.
附图说明Description of the drawings
图1为本公开实部分质粒示意图。Figure 1 is a schematic diagram of the actual part of the plasmid in this disclosure.
图2显示根据一个实施例的在293T细胞中通过瞬时转染的方法对比各转移载体质粒的产毒滴度。横坐标为各转移载体质粒和rtTA表达质粒组合，纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。Figure 2 shows the comparison of the toxin production titer of each transfer vector plasmid in 293T cells by transient transfection method according to an embodiment. The abscissa is the combination of each transfer vector plasmid and the rtTA expression plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
图3显示根据一个实施例的在稳定表达rtTA的293T细胞中通过瞬时转染的方法对比各转移载体质粒的产毒滴度。横坐标为各转移载体质粒所用的嵌合启动子或嵌合响应元件，纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。Figure 3 shows the comparison of the toxin production titer of each transfer vector plasmid by transient transfection method in 293T cells stably expressing rtTA according to an embodiment. The abscissa is the chimeric promoter or chimeric response element used in each transfer vector plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
图4显示根据一个实施例的在EuLV293T包装细胞中通过瞬时转染的方法对比各转移载体质粒的产毒滴度。横坐标为各转移载体质粒所用的嵌合启动子或嵌合响应元件，纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。Figure 4 shows the comparison of the toxin production titer of each transfer vector plasmid in EuLV293T packaging cells by transient transfection method according to an embodiment. The abscissa is the chimeric promoter or chimeric response element used in each transfer vector plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting virus transfection titer.
图5显示根据一个实施例的在慢病毒稳定生产细胞中各种调控病毒基因组转录盒转录的启动子或响应元件对产毒滴度的影响。横坐标为各慢病毒稳定生产细胞系中转录病毒基因组转录盒所用的嵌合启动子或嵌合响应元件，纵坐标为检测病毒转染滴度的Luciferase实验的RLU值。Figure 5 shows the effect of various promoters or response elements that regulate transcription of viral genome transcription cassettes on toxin production titer in a stable production cell of a lentivirus according to an embodiment. The abscissa is the chimeric promoter or chimeric response element used to transcribe the viral genome transcription cassette in each lentivirus stable production cell line, and the ordinate is the RLU value of the Luciferase experiment for detecting the virus transfection titer.
本发明的实施方式Embodiments of the present invention
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。The following examples are provided to illustrate the technical solutions of the present invention, and the following examples should not be considered as limiting the scope and spirit of the present invention.
实施例1：质粒构建方法Example 1: Plasmid construction method
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T4DNA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)；T4DNA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-10Gold，湖南丰晖生物科技有限公司，JZ011)；SEQ ID NO:1至SEQ ID NO:16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:20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：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, ligation and ligation of two or more DNA fragments by T4 DNA ligase The methods for product-competent cell transformation, plasmid preparation and identification in small quantities are all well-known techniques in the art. The following examples involve the following reagents: 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-10Gold, Hunan Fenghui Biotechnology Co., Ltd., JZ011); SEQ ID NO:1 to SEQ ID NO:16 shown by the nucleic acid sequence GenScript was synthesized and used in the construction of the plasmid described in the present disclosure, and the plasmid sequencing and identification were completed by Invitrogen. The schematic diagram of some plasmids used in the following examples is shown in Figure 1; Table 1 is the primer information for the plasmid construction; Table 2 is the description of the composition of the elements of the sequence SEQ ID NO: 1 to SEQ ID NO: 20; Table 3 is the plasmid Description of each functional element; Table 4 is the plasmid number and corresponding name constructed in this disclosure. The 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 by different codons, mutants of the above functional element sequences and gene sequences, and various functions The cloning position, cloning sequence and cloning direction of the element sequence and gene sequence. The specific plasmid construction method is as follows:
1.构建质粒18BF007和18BF004：将合成序列SEQ ID NO:2(2900bp)和SEQ ID NO:3(1386bp)用NotI和AsiSI酶切，并分别连接在质粒18BF003(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。1. Construction of plasmids 18BF007 and 18BF004: The synthetic sequences SEQ ID NO: 2 (2900bp) and SEQ ID NO: 3 (1386bp) were digested with NotI and AsiSI, and they were connected to plasmid 18BF003 (SEQ ID NO: 1,1893bp). The NotI and AsiSI restriction sites were used to construct plasmids 18BF007 and 18BF004, respectively.
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-BGI片段构建质粒18BF063。2. Construction of 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-BGI fragment to construct plasmid 18BF063.
3.构建质粒18BF072：以pRSV-Rev(Addgene，#12253)为模板、C-rev-F(SEQ ID NO:23)和C-rev-R(SEQ ID NO:24)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。3. Construction of plasmid 18BF072: using pRSV-Rev (Addgene, #12253) as template, C-rev-F (SEQ ID NO: 23) and C-rev-R (SEQ ID NO: 24) as primers for PCR amplification rev The gene fragment (380bp) was then digested with ClaI and XhoI and ligated to the ClaI and XhoI restriction sites of the 18BF063 plasmid to construct the 18BF072 plasmid.
4.构建质粒18BF068：以pMD2.G(Addgene，#12259)为模板，C-VSVG-F(SEQ ID NO:21)和C-VSVG-R(SEQ ID NO:22)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。4. Construction of plasmid 18BF068: use pMD2.G (Addgene, #12259) as template, C-VSVG-F (SEQ ID NO: 21) and C-VSVG-R (SEQ ID NO: 22) as primers for PCR amplification of VSV -G gene fragment (1565bp), then cut with ClaI and XhoI and ligated to the ClaI and XhoI sites of 18BF063 plasmid to construct plasmid 18BF068.
5.构建质粒18BF074和19BF126：以pMDLg/pRRE(Addgene，#12251)为模板、C-RRE-F(SEQ ID NO:25)和C-RRE-R(SEQ ID NO:26)为引物PCR扩增RRE基因片段(400bp)；C-GagPol-F(SEQ ID NO:27)和C-GagPol-R(SEQ ID NO:28)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18BF074质粒用BstBI酶切，凝胶回收8758bp(18BF074)片段并用T4连接酶连接从而构建得到质粒19BF126。5. Construction of plasmids 18BF074 and 19BF126: use pMDLg/pRRE (Addgene, #12251) as template, C-RRE-F (SEQ ID NO: 25) and C-RRE-R (SEQ ID NO: 26) as primers for PCR amplification Increase the RRE gene fragment (400bp); C-GagPol-F (SEQ ID NO: 27) and C-GagPol-R (SEQ ID NO: 28) are primers for PCR amplification of the gag/pol gene fragment (4336bp), and then use XbaI The two DNA fragments were digested with XhoI and EcoRI and XbaI respectively and ligated to the EcoRI and XhoI digestion sites of the 18BF007 plasmid to construct the 18BF074 plasmid. The 18BF074 plasmid was digested with BstBI, the 8758bp (18BF074) fragment was recovered from the gel and ligated with T4 ligase to construct the plasmid 19BF126.
6.构建质粒19BF257、19BF256和19BF075：将合成序列SEQ ID NO:7(633bp)和SEQ ID NO:8(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:9(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:10(768bp)和SEQ ID NO:11(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:8(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片段并将其连接在18BF085质粒的AvrII和AgeI酶切位点从而构建得到19BF075质粒。6. Construction of plasmids 19BF257, 19BF256 and 19BF075: The synthetic sequences SEQ ID NO: 7 (633 bp) and SEQ ID NO: 8 (1496 bp) were digested with ClaI and XhoI, SpeI and AgeI, respectively, and sequentially connected to ClaI of the 18BF007 plasmid And XhoI restriction sites and AvrII and AgeI restriction sites to construct the 19BF073 plasmid. The synthetic sequence SEQ ID NO: 9 (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 sequences SEQ ID NO: 10 (768 bp) and SEQ ID NO: 11 (765 bp) 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: 8 (1496 bp) 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 18BF085 plasmid to construct the 19BF075 plasmid.
7.构建质粒18BF019和18BF031：将合成序列SEQ ID NO:13(1044bp)和SEQ ID NO:12(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:14(1806bp)和SEQ ID NO:12(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。7. Construction of plasmids 18BF019 and 18BF031: The synthetic sequences SEQ ID NO: 13 (1044bp) and SEQ ID NO: 12 (1320 bp) were digested with BamHI and XhoI and XhoI and BglII, respectively, and ligated into the BamHI and BglII of the 18BF011 plasmid. The site thus constructed the 18BF019 plasmid. The synthetic sequences SEQ ID NO: 14 (1806 bp) and SEQ ID NO: 12 (1320 bp) were digested with BamHI and XhoI, and XhoI and BglII, respectively, and connected to the BamHI and BglII digestion sites of the 18BF011 plasmid to construct the 18BF031 plasmid.
8.构建质粒19BF081：以pRRLSIN.cPPT.PGK-GFP.WPRE(Addgene，#12252)为模板，hPGK-F(SEQ ID NO:29)和hPGK-R(SEQ ID NO:30)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:31)和Luc-R(SEQ ID NO:32)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序列从而构建得到18YYH26质粒。将合成序列SEQ ID NO:15(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:16(1320bp)用XhoI和BglII酶切并连接在19BF080质粒的XhoI和BamHI酶切位点从而构建得到19BF214质粒。将质粒18YYH26用PacI和XhoI酶切并分别凝胶回收DNA片段2272bp，并将回收的片段连接在19BF214质粒的PacI和XhoI酶切位点从而构建得到19BF081质粒。8. Construction of plasmid 19BF081: use pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252) as template, hPGK-F (SEQ ID NO: 29) and hPGK-R (SEQ ID NO: 30) as primers for PCR amplification Increase the PGK gene fragment (706bp); use pGL3-Basic (Promega, E1751) as a template, and Luc-F (SEQ ID NO: 31) and Luc-R (SEQ ID NO: 32) as primers to amplify the luciferase gene fragment ( 1728bp), after that, the two DNA fragments were digested with MluI and BamHI (538bp fragments recovered from the gel) and BamHI and XhoI, respectively, and connected to the MluI and XhoI sites of the 19BF126 plasmid, replacing the original plasmid DNA sequence to construct 18YYH26 Plasmid. The synthetic sequence SEQ ID NO: 15 (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: 16 (1320 bp) was digested with XhoI and BglII and connected to the XhoI and BamHI sites of the 19BF080 plasmid to construct the 19BF214 plasmid. Plasmid 18YYH26 was digested with PacI and XhoI, and the 2272bp DNA fragment was recovered by gel respectively, and the recovered fragment was ligated to the PacI and XhoI restriction sites of the 19BF214 plasmid to construct the 19BF081 plasmid.
9.构建质粒19BF123，19BF124，19BF125：以18BF007为模板，使用CMV(SpeI)-F(SEQ ID NO:33)和Fu-CMV-RU5-R(SEQ ID NO:35)为引物PCR扩增基因片段(585bp)，以合成序列SEQ ID NO:15(3610bp)为模板，使用Fu-CMV-RU5-F(SEQ ID NO:34)和GAG(ClaI)-R(SEQ ID NO:40)为引物扩增基因片段(725bp)，用融合PCR方法将这两段DNA连接并以CMV(SpeI)-F(SEQ ID NO:33)和GAG(ClaI)-R(SEQ ID NO:40)为引物PCR扩增得到基因片段(1282bp)，之后用SpeI和ClaI酶切处理并连接到19BF080质粒上的SpeI和CalI位点，构建质粒19BF120。以合成序列SEQ ID NO:6(315bp)为模板，使用TRE(SpeI)-F(SEQ ID NO:36)和Fu-CMV-RU5-R(SEQ ID NO:35)为引物PCR扩增基因片段(318bp)，以合成序列SEQ ID NO:15(3610bp)为模板，使用Fu-CMV-RU5-F(SEQ ID NO:34)和GAG(ClaI)-R(SEQ ID NO:40)为引物扩增基因片段(725bp)，用融合PCR方法将这两段DNA连接并以TRE(SpeI)-F(SEQ ID NO:36)和GAG(ClaI)-R(SEQ ID NO:40)为引物PCR扩增得到基因片段(1015bp)，之后用SpeI和ClaI酶切处理并连接到19BF080质粒上的SpeI和CalI位点，构建质粒19BF121。以合 成序列SEQ ID NO:5(302bp)为模板，使用TRE2(SpeI)-F(SEQ ID NO:39)和Fu_TRE2-RU5-R(SEQ ID NO:38)为引物PCR扩增基因片段(308bp)，以合成序列SEQ ID NO:15(3610bp)为模板，使用Fu-TRE2-RU5-F(SEQ ID NO:37)和GAG(ClaI)-R(SEQ ID NO:40)为引物扩增基因片段(725bp)，用融合PCR方法将这两段DNA连接并以TRE2(SpeI)-F(SEQ ID NO:39)和GAG(ClaI)-R(SEQ ID NO:40)为引物PCR扩增得到基因片段(1004bp)，之后用SpeI和ClaI酶切处理并连接到19BF080质粒上的SpeI和CalI位点，构建质粒19BF122。将质粒19BF081用PacI和PvuII酶切，凝胶回收基因片段4224bp，分别连接在质粒19BF120，19BF121和19BF122的PacI和PvuII酶切位点从而分别构建19BF123，19BF124和19BF125质粒。9. Construction of plasmids 19BF123, 19BF124, 19BF125: Using 18BF007 as a template, using CMV (SpeI)-F (SEQ ID NO: 33) and Fu-CMV-RU5-R (SEQ ID NO: 35) as primers for PCR amplification of genes Fragment (585bp), using synthetic sequence SEQ ID NO: 15 (3610 bp) as template, using Fu-CMV-RU5-F (SEQ ID NO: 34) and GAG (ClaI)-R (SEQ ID NO: 40) as primers Amplify the gene fragment (725bp), connect the two DNA fragments by fusion PCR method and use CMV(SpeI)-F(SEQ ID NO:33) and GAG(ClaI)-R(SEQ ID NO:40) as primers for PCR The amplified gene fragment (1282bp) was then digested with SpeI and ClaI and ligated to the SpeI and CalI sites on the 19BF080 plasmid to construct the plasmid 19BF120. Using the synthetic sequence SEQ ID NO: 6 (315bp) as a template, using TRE (SpeI)-F (SEQ ID NO: 36) and Fu-CMV-RU5-R (SEQ ID NO: 35) as primers for PCR amplification of gene fragments (318bp), using the synthetic sequence SEQ ID NO: 15 (3610 bp) as a template, using Fu-CMV-RU5-F (SEQ ID NO: 34) and GAG (ClaI)-R (SEQ ID NO: 40) as primers. To increase the gene fragment (725bp), use the fusion PCR method to connect the two DNA fragments and use TRE(SpeI)-F(SEQ ID NO:36) and GAG(ClaI)-R(SEQ ID NO:40) as primers for PCR amplification. The gene fragment (1015bp) was increased, and then digested with SpeI and ClaI and ligated to the SpeI and CalI sites on the 19BF080 plasmid to construct the plasmid 19BF121. Using the synthetic sequence SEQ ID NO: 5 (302 bp) as a template, using TRE2 (SpeI)-F (SEQ ID NO: 39) and Fu_TRE2-RU5-R (SEQ ID NO: 38) as primers PCR amplified gene fragments (308 bp) ), using the synthetic sequence SEQ ID NO: 15 (3610 bp) as a template, using Fu-TRE2-RU5-F (SEQ ID NO: 37) and GAG (ClaI)-R (SEQ ID NO: 40) as primers to amplify the gene Fragment (725bp), the two DNA fragments were joined by fusion PCR method and PCR amplified with TRE2(SpeI)-F(SEQ ID NO:39) and GAG(ClaI)-R(SEQ ID NO:40) as primers The gene fragment (1004bp) was then digested with SpeI and ClaI and ligated to the SpeI and CalI sites on the 19BF080 plasmid to construct plasmid 19BF122. Plasmid 19BF081 was digested with PacI and PvuII, and the 4224bp gene fragment was recovered from the gel and ligated to the PacI and PvuII sites of plasmids 19BF120, 19BF121 and 19BF122 respectively to construct 19BF123, 19BF124 and 19BF125 plasmids, respectively.
表1.引物信息列表Table 1. Primer information list
Figure PCTCN2020115521-appb-000002
Figure PCTCN2020115521-appb-000002
表2.序列元件组成说明Table 2. Composition description of sequence elements
Figure PCTCN2020115521-appb-000003
Figure PCTCN2020115521-appb-000003
Figure PCTCN2020115521-appb-000004
Figure PCTCN2020115521-appb-000004
表3.质粒功能元件说明Table 3. Description of plasmid functional elements
Figure PCTCN2020115521-appb-000005
Figure PCTCN2020115521-appb-000005
Figure PCTCN2020115521-appb-000006
Figure PCTCN2020115521-appb-000006
表4.质粒编号及名称Table 4. Plasmid number and name
质粒编号Plasmid number 质粒名称Plasmid name
18BF00318BF003 pma-MCSpma-MCS
18BF00718BF007 pmaSBT3-2xHS4I-CMV-BGI-MCSpmaSBT3-2xHS4I-CMV-BGI-MCS
18BF00418BF004 pmaHPBT-2xHS4I-MCSpmaHPBT-2xHS4I-MCS
18BF01118BF011 pmaCMV-BGI-MCSpmaCMV-BGI-MCS
18BF06318BF063 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-MCS pmaSBT3-2xHS4I-TRE 3G CuO-BGI-MCS
18BF07218BF072 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-rev pmaSBT3-2xHS4I-TRE 3G CuO-BGI-rev
18BF06818BF068 pmaSBT3-2xHS4I-TRE 3GCuO-BGI-VSVG pmaSBT3-2xHS4I-TRE 3G CuO-BGI-VSVG
18BF07418BF074 pmaSBT3-2xHS4I-CMV-BGI-gag/pol-RREpmaSBT3-2xHS4I-CMV-BGI-gag/pol-RRE
19BF12619BF126 pmaSBT3-2xHS4I-CMV-gag/pol-RREpmaSBT3-2xHS4I-CMV-gag/pol-RRE
19BF07319BF073 pmaSBT3-2xHS4I-CMV-BGI-optiCymR-optiHygroRpmaSBT3-2xHS4I-CMV-BGI-optiCymR-optiHygroR
18BF00818BF008 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-MCSpmaSBT3-2xHS4I-CAGGS-BGI(C&R)-MCS
18BF08518BF085 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G
18BF08418BF084 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv
19BF25719BF257 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G-optiHygroR pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G -optiHygroR
19BF25619BF256 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv-optiHygroR pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-rtTA adv -optiHygroR
19BF07519BF075 pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G-CMV-BGI-optiCymR-optiHygroR pmaSBT3-2xHS4I-CAGGS-BGI(C&R)-optirtTA 3G -CMV-BGI-optiCymR-optiHygroR
18BF01918BF019 pmaCMV-BGI-SB-IRES-ECFPpmaCMV-BGI-SB-IRES-ECFP
18BF03118BF031 pmaCMV-BGI-PB-IRES-ECFPpmaCMV-BGI-PB-IRES-ECFP
18YYH2618YYH26 pmaSBT3-2xHS4I-hPGK-LucpmaSBT3-2xHS4I-hPGK-Luc
19BF08019BF080 pmaHPBT-2xHS4I-LVRSV-RU5-MCS-optiPuroRpmaHPBT-2xHS4I-LVRSV-RU5-MCS-optiPuroR
19BF21419BF214 pmaHPBT-2xHS4I-LVRSV-RU5-MCS-IRES-EGFP-optiPuroRpmaHPBT-2xHS4I-LVRSV-RU5-MCS-IRES-EGFP-optiPuroR
19BF08119BF081 pmaHPBT-2xHS4I-LVRSV-RU5-hPGK-Luc-IRES-EGFP-optiPuroRpmaHPBT-2xHS4I-LVRSV-RU5-hPGK-Luc-IRES-EGFP-optiPuroR
19BF12019BF120 pmaHPBT-2xHS4I-LVCMV-RU5-MCS-optiPuroRpmaHPBT-2xHS4I-LVCMV-RU5-MCS-optiPuroR
19BF12119BF121 pmaHPBT-2xHS4I-LVTRE1-RU5-MCS-optiPuroRpmaHPBT-2xHS4I-LVTRE1-RU5-MCS-optiPuroR
19BF12219BF122 pmaHPBT-2xHS4I-LVTRE2-RU5-MCS-optiPuroRpmaHPBT-2xHS4I-LVTRE2-RU5-MCS-optiPuroR
19BF12319BF123 pmaHPBT-2xHS4I-LVCMV-RU5-hPGK-Luc-IRES-EGFP-optiPuroRpmaHPBT-2xHS4I-LVCMV-RU5-hPGK-Luc-IRES-EGFP-optiPuroR
19BF12419BF124 pmaHPBT-2xHS4I-LVTRE1-RU5-hPGK-Luc-IRES-EGFP-optiPuroRpmaHPBT-2xHS4I-LVTRE1-RU5-hPGK-Luc-IRES-EGFP-optiPuroR
19BF12519BF125 pmaHPBT-2xHS4I-LVTRE2-RU5-hPGK-Luc-IRES-EGFP-optiPuroRpmaHPBT-2xHS4I-LVTRE2-RU5-hPGK-Luc-IRES-EGFP-optiPuroR
实施例2：通过在293T细胞中瞬时转染Tet响应元件调控的病毒基因组转录盒构建体制备慢病毒载体Example 2: Preparation of a lentiviral vector by transiently transfecting a Tet response element-regulated viral genome transcription cassette construct in 293T cells
本实施例通过将慢病毒包装所需基因rev、VSV-G、gag/pol和Tet-On诱导表达系统的反式激活物rtTA adv或rtTA 3G的编码序列以及本公开所述受Tet响应元件调控的携带目的核酸片段的病毒基因组转录盒的构建体(转移载体质粒)瞬时转染293T细胞的方法生产慢病毒，并比较本公开所述受Tet响应元件调控的病毒基因组转录盒对比使用组成型活性启动子调控的病毒基因组转录盒的产毒滴度。本实施例共验证4种转移载体质粒19BF081(组成型活性启动子RSV-RU5)、19BF123(组成型活性启动子CMV-RU5)、19BF124(TRE1-RU5)和19BF125(TRE2-RU5)，分别以使用RSV或CMV组成型活性启动子，或本公开所述的TRE1或TRE2Tet响应元件调控携带目的转基因的慢病毒基因组表达盒的转录。本实施例以hPGK-Luciferase-IRES-EGFP序列为目的核酸片段，并基于Luciferase酶活性对比各转移载体质粒在瞬时生产条件下的产毒滴度。具体实验方法如下。 In this embodiment, the coding sequence of the transactivator rtTA adv or rtTA 3G of the transactivator rtTA adv or rtTA 3G of the expression system is induced by the genes required for lentivirus packaging, rev, VSV-G, gag/pol and Tet-On, and regulated by the Tet response element described in the present disclosure. The method of transiently transfecting 293T cells with the construct of the viral genome transcription cassette carrying the target nucleic acid fragment (transfer vector plasmid) produces lentivirus, and compares the viral genome transcription cassette regulated by the Tet response element described in the present disclosure and contrasts the use of constitutive activity The toxin production titer of the viral genome transcription cassette regulated by the promoter. This example verifies 4 kinds of transfer vector plasmids 19BF081 (constitutively active promoter RSV-RU5), 19BF123 (constitutively active promoter CMV-RU5), 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5), respectively The RSV or CMV constitutively active promoter, or the TRE1 or TRE2Tet response element described in the present disclosure is used to regulate the transcription of the lentiviral genome expression cassette carrying the transgene of interest. In this example, the hPGK-Luciferase-IRES-EGFP sequence was used as the target nucleic acid fragment, and the toxin production titer of each transfer vector plasmid under transient production conditions was compared based on the Luciferase enzyme activity. The specific experimental method is as follows.
将293T细胞按1.5E+05个细胞每孔接种于24孔板中，培养在37℃、5％CO 2环境下，培养体积为500μL含有10％FBS(ExCell，11H116)的DMEM(Sigam，D6429)培养基。接种24小时后，按照PEI方法转染细胞，操作方法如下：转染时每孔加入50μL转染试剂，其中含有0.8μg的总质粒量和3.2μg的PEI MAX(Polysciences，24765-1)，其中质粒摩尔比pMD2.G(Addgene 12259):pMDLg/PRRE(Addgene 12251):pRSV-Rev(Addgene12253):rtTA(19BF257或19BF256)：转移载体质粒为1:1:1:1:1。其中转移载体为19BF081、19BF123、19BF124和19BF125；Tet-On反式激活物编码质粒为19BF257(rtTA 3G)或19BF256(rtTA adv)，每种样品设置复孔。转染3小时后，更换培养基并在每种样品其中一个复孔中加入诱导剂(2mmol/L丁酸钠(Sigma，303410)，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))，另外一个复孔仅加入2mmol/L丁酸钠。继续培养48小时后，以4500rpm离心15分钟方法收集病毒上清液。 Inoculate 293T cells in a 24-well plate with 1.5E+05 cells per well, culture at 37°C and 5% CO 2 in a culture volume of 500 μL DMEM (Sigam, D6429) containing 10% FBS (ExCell, 11H116) ) Medium. After 24 hours of inoculation, cells were transfected according to the PEI method. The operation method is as follows: during transfection, add 50μL of transfection reagent to each well, which contains 0.8μg of total plasmid and 3.2μg of PEI MAX (Polysciences, 24765-1). The molar ratio of plasmid pMD2.G (Addgene 12259): pMDLg/PRRE (Addgene 12251): pRSV-Rev (Addgene 12253): rtTA (19BF257 or 19BF256): the transfer vector plasmid is 1:1:1:1:1. The transfer vectors are 19BF081, 19BF123, 19BF124, and 19BF125; the Tet-On transactivator encoding plasmid is 19BF257 (rtTA 3G ) or 19BF256 (rtTA adv ), and multiple wells are set for each sample. After 3 hours of transfection, change the medium and add an inducer (2mmol/L sodium butyrate (Sigma, 303410), 1μg/ml DOX (doxycycline hydrochloride (DOX), raw material) to one of the multiple wells of each sample. Industrial Biological Engineering (Shanghai) Co., Ltd., A600889)), only 2mmol/L sodium butyrate was added to the other multiple hole. After culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes.
使用HT1080细胞Luciferase病毒滴度检测方法检测各样品的产毒滴度，具体操作如下：诱导产毒24小时后，将HT1080细胞按照1E+04个细胞每孔接种于96孔板(Corning 3916)中，培养基为DMEM完全培养基。在检测滴度前1小时，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。之后将上述离心后所得病毒上清液按每种样品三个孔每孔50μl加入96孔板中，阴性对照孔加入50μL DMEM完全培养基。培养48小时后用
Figure PCTCN2020115521-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)。检测结果如图1所示。 The HT1080 cell Luciferase virus titer detection method was used to detect the toxin production titer of each sample. The specific operation is as follows: 24 hours after the induction of toxin production, HT1080 cells were seeded in a 96-well plate (Corning 3916) with 1E+04 cells per well , The medium is DMEM complete medium. One hour before the detection of titer, the medium of HT1080 cells was replaced with DMEM complete medium containing 8 μg/ml polybrene (Sigam, H9268). Then, the virus supernatant obtained after the above centrifugation was added to a 96-well plate with 50 μl of each sample in three wells, and 50 μL of DMEM complete medium was added to the negative control well. Use after 48 hours incubation
Figure PCTCN2020115521-appb-000007
Luciferase Assay System (Promega, E2610) kit, according to its operating instructions (Promega, FB037) to detect the relative light unit RLU (relative light unit) of each well, the detection instrument is a fluorescence microplate reader (Perkin Elmer Victor V). The test results are shown in Figure 1.
图2结果显示，在使用瞬时转染293T细胞的方法包装慢病毒时，使用TRE1-RU5(19BF124)和TRE2-RU5(19BF125)Tet嵌合响应元件调控慢病毒基因组表达盒转录的转移载体质粒的产毒滴度显著高于传统使用组成型活性启动子的转移载体质粒。使用TRE1-RU5嵌合响应元件的19BF124质粒在搭配rtTA adv反式激活物时，产毒滴度分别是RSV-RU5或CMV-RU5的2.93倍或2.33倍；在搭配rtTA 3G时，产毒滴度分别是RSV-RU5或CMV-RU5的2.55倍或2.02倍。使用TRE2-RU5嵌合响应元件的19BF125质粒在搭配rtTA adv反式激活物时，产毒滴度分别是RSV-RU5或CMV-RU5的1.75倍或1.39倍；在搭配rtTA 3G时，产毒滴度分别是RSV-RU5或CMV-RU5的1.83倍或1.45倍。 Figure 2 shows that when the lentivirus is packaged by transiently transfecting 293T cells, the TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) Tet chimeric response elements are used to regulate the transcription of the transfer vector plasmid of the lentiviral genome expression cassette. The toxin production titer is significantly higher than that of traditional transfer vector plasmids using constitutively active promoters. When the 19BF124 plasmid with TRE1-RU5 chimeric response element is matched with rtTA adv transactivator, the toxin production titer is 2.93 times or 2.33 times that of RSV-RU5 or CMV-RU5 , respectively; when matched with rtTA 3G , it produces toxin drops The degree is 2.55 times or 2.02 times that of RSV-RU5 or CMV-RU5, respectively. When the 19BF125 plasmid using the TRE2-RU5 chimeric response element is matched with the rtTA adv transactivator, the toxin production titers are 1.75 times or 1.39 times that of RSV-RU5 or CMV-RU5 , respectively; when matched with rtTA 3G , the toxin production drops The degree is 1.83 times or 1.45 times that of RSV-RU5 or CMV-RU5, respectively.
实施例3：通过在稳定表达rtTA的293T细胞中瞬时转染Tet响应元件调控的病毒基因组转录盒构建体制备慢病毒载体Example 3: Preparation of a lentiviral vector by transiently transfecting a Tet response element-regulated viral genome transcription cassette construct in 293T cells stably expressing rtTA
本实施例通过将慢病毒包装所需基因rev、VSV-G、gag/pol和本公开所述受Tet响应元件调控的携带目的核酸片段的病毒基因组转录盒的构建体(转移载体质粒)瞬时转染稳定表达反式激活物rtTA的293T细胞的方法生产慢病毒，并比较本公开所述受Tet响应元件调控的病毒基因组转录盒对比使用组成型活性启动子调控的病毒基因组转录盒的产毒滴度。本实施例所用转移载体质粒和实施例2中所述一致，分别为19BF081、19BF123、19BF124和19BF125。具体实验方法如下。In this example, the genes rev, VSV-G, gag/pol required for lentivirus packaging and the constructs (transfer vector plasmids) of the viral genome transcription cassettes (transfer vector plasmids) that are regulated by the Tet response elements of the present disclosure and are regulated by the Tet response element The method of transfecting 293T cells stably expressing the transactivator rtTA to produce lentivirus, and comparing the viral genome transcription cassette regulated by the Tet response element described in the present disclosure to the viral genome transcription cassette regulated by a constitutively active promoter Spend. The transfer vector plasmid used in this example is the same as that described in Example 2, and they are 19BF081, 19BF123, 19BF124, and 19BF125, respectively. The specific experimental method is as follows.
1.通过“睡美人(Sleeping Beauty，简称SB)”转座子系统构建293T-rtTA adv和293T-rtTA 3G细胞系： 1. Construct 293T-rtTA adv and 293T-rtTA 3G cell lines through the "Sleeping Beauty (SB)" transposon system:
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5.5ug，PEI和质粒质量比为4：1，其中按照质粒19BF256:18BF019(表达SB转座酶)摩尔比10:1进行转染以获得293T-rtTA adv细胞；按照质粒19BF257:18BF019摩尔比10:1进行转染以获得293T-rtTA 3G细胞。转染后用潮霉素(Hygromycin，生工A600230-0001)药物筛选至少三代后，细胞在药物筛选压力下生长和原始293T细胞一致后，进行以下实验。 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 24 hours of culture, transfection was carried out according to the PEI method. The total plasmid amount was 5.5ug, and the mass ratio of PEI to plasmid was 4:1. The molar ratio of plasmid 19BF256:18BF019 (expressing SB transposase) was 10:1 for transfection. Obtain 293T-rtTA adv cells; according to plasmid 19BF257:18BF019 molar ratio of 10:1 for transfection to obtain 293T-rtTA 3G cells. After transfection with hygromycin (Hygromycin, Shenggong A600230-0001) drug screening for at least three generations, the cells grew under the pressure of drug screening to be consistent with the original 293T cells, and then the following experiment was performed.
2.在稳定表达rtTA的293T细胞中通过瞬时转染的方法对比各转移载体质粒的产毒滴度2. In 293T cells stably expressing rtTA, the toxin production titer of each transfer vector plasmid was compared by transient transfection method
将293T-rtTA adv和293T-rtTA 3G两种细胞系分别按照1.5E+05个细胞每孔接种于24孔板中，培养体积为500μL DMEM完全培养基。接种24小时后，按照PEI方法转染细胞，操作方法如下：转染时每孔加入50μL转染试剂，其中含有0.8μg的总质粒量和3.2μg的PEI MAX(Polysciences，24765-1)，其中摩尔比pMD2.G(Addgene 12259):pMDLg/PRRE(Addgene 12251):pRSV-Rev(Addgene 12253):转移载体质粒为1:1:1:1。其中转移载体为19BF081(RSV-RU5)、19BF123(CMV-RU5)、19BF124(TRE1-RU5)或19BF125(TRE2-RU5)，每种样品设置复孔。转染3小时后，更换培养基，并在每种样品其中一个复孔加入诱导剂(2mmol/L丁酸钠，1μg/ml DOX)，另外一个复孔仅加入2mmol/L丁酸钠。继续培养48小时后，以4500rpm离心15分钟方法收集病毒上清液。进行HT1080细胞Luciferase病毒滴度检测，检测方法同实施例2一致，结果如图3所示。 Two cell lines, 293T-rtTA adv and 293T-rtTA 3G , were seeded in a 24-well plate with 1.5E+05 cells per well, and the culture volume was 500 μL DMEM complete medium. After 24 hours of inoculation, cells were transfected according to the PEI method. The operation method is as follows: during transfection, add 50μL of transfection reagent to each well, which contains 0.8μg of total plasmid and 3.2μg of PEI MAX (Polysciences, 24765-1). The molar ratio pMD2.G (Addgene 12259): pMDLg/PRRE (Addgene 12251): pRSV-Rev (Addgene 12253): transfer vector plasmid is 1:1:1:1. Among them, the transfer vector is 19BF081 (RSV-RU5), 19BF123 (CMV-RU5), 19BF124 (TRE1-RU5) or 19BF125 (TRE2-RU5), and multiple holes are set for each sample. After 3 hours of transfection, the medium was changed, and an inducer (2mmol/L sodium butyrate, 1μg/ml DOX) was added to one of the multiple wells of each sample, and only 2mmol/L sodium butyrate was added to the other multiple well. After culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes. The Luciferase virus titer of HT1080 cells was detected, and the detection method was the same as that in Example 2, and the result is shown in FIG. 3.
图3结果所示，使用TRE1-RU5(19BF124)和TRE2-RU5(19BF125)嵌合响应元件调控慢病毒基因组表达盒转录的转移载体质粒在DOX诱导下产毒滴度明显高于传统使用组成型活性启动子RSV和CMV的转移载体质粒。在293T-rtTA adv细胞中，19BF124(TRE1-RU5)和19BF125(TRE2-RU5)的诱导产毒滴度分别为1.57E+06RLU和1.38E+06RLU；分别为19BF081(RSV-RU5)转移载体质粒的6.7倍和5.9倍，分别为19BF123(CMV-RU5)转移载体质粒的2.48倍和2.18倍。在293T-rtTA 3G细胞中，19BF124(TRE1-RU5)和19BF125(TRE2-RU5)的诱导产毒滴度分别为2.36E+06RLU和1.97E+6RLU；分别为19BF081(RSV-RU5)转移载体质粒的7.7倍和6.5倍，分别为19BF123(CMV-RU5)转移载体质粒的2.4倍和2.0倍。在非诱导条件下，转染19BF124(TRE1-RU5)和19BF125(TRE2-RU5)转移载体质粒在293T-rtTA adv和293T-rtTA 3G细胞中无明显产毒滴度，证明TRE1-RU5和TRE2-RU5嵌合Tet响应元件仅在有诱导剂条件下才能转录慢病毒基因组RNA并包装病毒。 The results in Figure 3 show that the transfer vector plasmid that uses TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) chimeric response elements to regulate the transcription of the lentiviral genome expression cassette is significantly higher than the traditionally used constitutive type under DOX induction. Active promoter RSV and CMV transfer vector plasmid. In 293T-rtTA adv cells, the induced toxin production titers of 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5) were 1.57E+06RLU and 1.38E+06RLU, respectively; they were 19BF081 (RSV-RU5) transfer vector plasmid 6.7 times and 5.9 times of the transfer vector plasmid of 19BF123 (CMV-RU5), 2.48 times and 2.18 times, respectively. In 293T-rtTA 3G cells, the induced toxin production titers of 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5) were 2.36E+06RLU and 1.97E+6RLU, respectively; they were 19BF081 (RSV-RU5) transfer vector plasmids, respectively 7.7 times and 6.5 times, respectively, 2.4 times and 2.0 times of the transfer vector plasmid of 19BF123 (CMV-RU5). Under non-inducing conditions, the transfection of 19BF124 (TRE1-RU5) and 19BF125 (TRE2-RU5) transfer vector plasmids has no obvious toxin production titers in 293T-rtTA adv and 293T-rtTA 3G cells, which proves that TRE1-RU5 and TRE2- The RU5 chimeric Tet response element can only transcribe lentiviral genomic RNA and package the virus in the presence of an inducer.
实施例4：通过在稳定表达慢病毒包装基因的EuLV293T包装细胞中瞬时转染Tet响应元件调控的病毒基因组转录盒构建体制备慢病毒载体Example 4: Preparation of a lentiviral vector by transiently transfecting a Tet response element-regulated viral genome transcription cassette construct in EuLV293T packaging cells stably expressing lentiviral packaging genes
本实施例先通过“睡美人(Sleeping Beauty，简称SB)”转座子系统构建在293T基因组中稳定整合慢病毒包装所需基因rev、VSV-G和gag/pol以及Tet-On诱导表达系统中的rtTA 3G反式激活物和Cumate诱导表达系统的CymR阻遏物的EuLV293T稳定包装细胞系。其中rev基因和VSV-G基因的表达受Tet-On和Cumate复合诱导表达系统调控，用于构建细胞系的质粒为18BF072和18BF068；gag/pol基因的表达受CMV启动子调控，用于构建细胞系的质粒为18BF074；反式激活物rtTA 3G和阻遏物CymR蛋白的表达分别受CAGGS启动子和CMV启动子调控并且两个蛋白的表达盒(expression cassette)被共同构建在19BF075质粒上。之后通过将本公开所述受Tet响应元件调控的携带目的核酸片段的病毒基因组转录盒的构建体(转移载体质粒)瞬时转染上述EuLV293T包装细胞中，并比较本公开所述受Tet响应元件调控的病毒基因组转录盒对比使用组成型活性启动子调控的病毒基因组转录盒的产毒滴度。本实施例所用转移载体质粒和实施例2中所述一致，分别为 19BF081、19BF123、19BF124和19BF125。具体实验方法如下： In this example, the "Sleeping Beauty (SB)" transposon system was first constructed to stably integrate the genes rev, VSV-G and gag/pol required for lentivirus packaging in the 293T genome, as well as the Tet-On inducible expression system The EuLV293T stable packaging cell line of the rtTA 3G transactivator and Cumate inducible expression system of the CymR repressor. The expression of rev gene and VSV-G gene is regulated by the Tet-On and Cumate compound induction expression system. The plasmids used to construct the cell line are 18BF072 and 18BF068; the expression of gag/pol gene is regulated by the CMV promoter and is used to construct cells The plasmid of the line is 18BF074; the expression of the transactivator rtTA 3G and the repressor CymR protein are regulated by the CAGGS promoter and CMV promoter, respectively, and the expression cassettes of the two proteins are jointly constructed on the 19BF075 plasmid. Then, the construct (transfer vector plasmid) of the viral genome transcription cassette carrying the target nucleic acid fragment regulated by the Tet response element of the present disclosure was transiently transfected into the above-mentioned EuLV293T packaging cell, and compared with the regulation of the Tet response element of the present disclosure The viral genome transcription cassette was compared with the toxin production titer of the viral genome transcription cassette regulated by a constitutively active promoter. The transfer vector plasmid used in this example is the same as that described in Example 2, and they are 19BF081, 19BF123, 19BF124, and 19BF125, respectively. The specific experimental methods are as follows:
1.通过SB转座子系统构建EuLV293T慢病毒包装细胞系1. Construction of EuLV293T lentiviral packaging cell line through SB transposon system
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和实施例3。将293T细胞(ATCC，CRL3216)按1.5E+06细胞接种于60mm培养皿中，培养基为3ml DMEM完全培养基(DMEM(Sigma，D6429)添加10％FBS(ExCell，11H116))，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19BF075:19BF72:18BF068:19BF74:18BF019摩尔比为3:3:2:12:2进行转染。转染后24小时，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。维持此筛选压力传代培养至细胞稳定生长，判断依据为：(1)细胞生长速度与原始293T细胞相同，(2)SB表达质粒18BF019基本消失，表现为ECFP阳性细胞比例跌至1％以下，(3)死亡细胞比例小于5％。最终得到在基因组稳定整合rtTA 3G、CymR、rev、VSV-G和gag/pol蛋白编码序列的慢病毒包装细胞系，并将其命名为EuLV293T。 Refer to Example 2 and Example 3 for the experimental procedures of cell culture, PEI transfection, and hygromycin screening. Inoculate 293T cells (ATCC, CRL3216) according to 1.5E+06 cells in a 60mm culture dish with 3ml DMEM complete medium (DMEM (Sigma, D6429) supplemented with 10% FBS (ExCell, 11H116)) at 37°C Cultivate for 24 hours under the condition of 5% CO 2. Plasmid transfection was carried out by the PEI transfection method, the total plasmid amount was 5μg, the mass ratio of PEI to total plasmid was 4:1, and the molar ratio of plasmid 19BF075:19BF72:18BF068:19BF74:18BF019 was 3:3:2:12: 2 Perform transfection. Twenty-four hours after transfection, all cells were inoculated into a 100mm petri dish and continued to be cultured, and 200μg/ml hygromycin (Sangong A600230-0001) was added for selection. Maintain this selection pressure and subculture until the cells grow stably. The judgment is based on: (1) The cell growth rate is the same as that of the original 293T cells, (2) The SB expression plasmid 18BF019 basically disappears, showing that the proportion of ECFP-positive cells has fallen below 1%, ( 3) The proportion of dead cells is less than 5%. Finally, a lentiviral packaging cell line with stable integration of rtTA 3G , CymR, rev, VSV-G and gag/pol protein coding sequences in the genome was obtained, and it was named EuLV293T.
2.在EuLV293T包装细胞中通过瞬时转染的方法对比各转移载体质粒的产毒滴度2. Compare the toxin production titer of each transfer vector plasmid in EuLV293T packaging cells by transient transfection method
将EuLV293T细胞培养在37℃、5％CO 2环境下，培养基为含有10％FBS(ExCell，11H116)的DMEM(Sigam，D6429)培养基。将细胞系按照1.5E+05个细胞每孔接种于24孔板中，培养体积为500μL。转染时每孔加入50μL转染试剂，其中含有0.26μg转移载体质粒、0.54μg18BF003质粒和3.2μg的PEI MAX(Polysciences，24765-1)，每个条件设复孔。转染3h后，更换培养基为含有5mmol/L丁酸钠的完全培养基，并在每个样品复孔的其中一个孔中加入1μg/ml DOX和200μg/ml Cumate(Aladdin，I107765)，设为诱导组；另一孔加入等量培养基，设为非诱导组。继续培养48小时后，以4500rpm离心15分钟方法收集病毒上清液。进行HT1080细胞Luciferase病毒滴度检测，检测方法同实施例2一致，结果如图4所示。 EuLV293T cells were cultured at 37°C and 5% CO 2 in a DMEM (Sigam, D6429) medium containing 10% FBS (ExCell, 11H116). The cell line was seeded in a 24-well plate with 1.5E+05 cells per well, and the culture volume was 500 μL. During transfection, 50μL of transfection reagent was added to each well, which contained 0.26μg of transfer vector plasmid, 0.54μg of 18BF003 plasmid and 3.2μg of PEI MAX (Polysciences, 24765-1), and each condition was equipped with multiple wells. After 3 hours of transfection, change the medium to a complete medium containing 5mmol/L sodium butyrate, and add 1μg/ml DOX and 200μg/ml Cumate (Aladdin, I107765) to one of the wells of each sample. It is the induction group; another well is added with the same amount of medium to make it the non-induction group. After culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes. The Luciferase virus titer of HT1080 cells was detected, and the detection method was the same as that in Example 2, and the result is shown in FIG. 4.
图4结果所示，使用TRE1-RU5(19BF124)和TRE2-RU5(19BF125)Tet嵌合响应元件调控慢病毒基因组表达盒转录的转移载体质粒在加入DOX和Cumate诱导下产毒滴度明显高于传统使用组成型活性启动子RSV和CMV的转移载体质粒。TRE1-RU5(19BF124)和TRE2-RU5(19BF125)的诱导产毒滴度分别为2.42E+06RLU和2.14E+06RLU；分别为19BF081(RSV-RU5)转移载体质粒的3.4倍和3.0倍，分别为19BF123(CMV-RU5)转移载体质粒的1.6倍和1.4倍。在非诱导条件下，所有转移载体质粒在EuLV293T细胞中无明显查毒滴度，证明EuLV293T细胞仅在有诱导剂条件下才能转录慢病毒基因组并且包装慢病毒。The results in Figure 4 show that the transfer vector plasmid that uses TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) Tet chimeric response elements to regulate the transcription of the lentiviral genome expression cassette has significantly higher toxin production titers under the induction of DOX and Cumate The traditional use of constitutively active promoters RSV and CMV transfer vector plasmids. The induced toxin production titers of TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) were 2.42E+06RLU and 2.14E+06RLU, respectively; they were 3.4 times and 3.0 times that of the transfer vector plasmid of 19BF081 (RSV-RU5), respectively It is 1.6 times and 1.4 times of the transfer vector plasmid of 19BF123 (CMV-RU5). Under non-inducing conditions, all the transfer vector plasmids have no obvious virus titers in EuLV293T cells, which proves that EuLV293T cells can transcribe the lentiviral genome and package lentivirus only under the condition of inducer.
实施例5：通过Tet响应元件调控的病毒基因组转录盒在慢病毒稳定生产细胞系中制备慢病毒Example 5: Preparation of lentivirus in a stable production cell line of lentivirus by a viral genome transcription cassette regulated by Tet response elements
本实施例首先通过来源于粉纹夜蛾(Trichoplusia ni)的“PiggyBac(PB)转座子系统将被不同启动子或Tet响应元件调控转录的携带hPGK-Luciferase-EGFP目的核酸片段的病毒基因组转录盒稳定整合在实施例4中制备的EuLV293T包装细胞的基因组中，其中用于构建慢病毒生产细胞系的转移载体质粒分别为：19BF081、19BF123、19BF124和19BF125。之后再通过诱导的方式制备慢病毒，并比较本公开所述受Tet响应元件调控的病毒基因组转录盒对比使用组成型活性启动子调控的病毒基因组转录盒的产毒滴度。具体实验方法如下：In this example, the "PiggyBac (PB) transposon system derived from Trichoplusia ni) was used to transcribe viral genomes carrying hPGK-Luciferase-EGFP target nucleic acid fragments that are regulated by different promoters or Tet response elements. The cassette is stably integrated into the genome of the EuLV293T packaging cell prepared in Example 4. The transfer vector plasmids used to construct the lentivirus production cell line are: 19BF081, 19BF123, 19BF124 and 19BF125. Then the lentivirus is prepared by induction. , And compare the toxin production titer of the viral genome transcription cassette regulated by the Tet response element described in the present disclosure and the viral genome transcription cassette regulated by a constitutive active promoter. The specific experimental methods are as follows:
1.通过PB转座子系统构建慢病毒稳定生产细胞系1. Construct a stable lentivirus production cell line through the PB transposon system
将EuLV293T细胞按1.5E+06细胞每孔接种于60mm培养皿中，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5.5ug，其中按照转移载体质粒:18BF031(表达PB转座酶)摩尔比10:1进行转染，PEI总量为22ug。其中转移载体质粒为19BF081、19BF123、19BF124或19BF125。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代至细胞系稳定，得到4条慢病毒稳转生产细胞系，分别为EuLV293T-19BF081、EuLV293T-19BF123、EuLV293T-19BF124和EuLV293T-19BF125。 EuLV293T cells were seeded in a 60mm culture dish with 1.5E+06 cells per well. The medium was 3ml DMEM complete medium. After culturing for 24 hours at 37°C under 5% CO 2 conditions, transfection was carried out according to the PEI method. The total plasmid The amount is 5.5ug, of which the transfer vector plasmid: 18BF031 (expressing PB transposase) molar ratio is 10:1 for transfection, and the total amount of PEI is 22ug. The transfer vector plasmid is 19BF081, 19BF123, 19BF124 or 19BF125. 24 hours after transfection, 2.5μg/ml puromycin (Puromycin, Aladdin P113126) was added to screen for at least 3 generations until the cell line was stable, and 4 stable lentivirus production cell lines were obtained, namely EuLV293T-19BF081, EuLV293T-19BF123, EuLV293T-19BF124 and EuLV293T-19BF125.
2.使用不同转移载体质粒构建的慢病毒稳定生产细胞系的产毒能力对比2. Comparison of toxin production capacity of lentivirus stable production cell lines constructed using different transfer vector plasmids
将上述四种慢病毒稳定生产细胞按1.5E+05个细胞每孔接种于24孔板中，培养体积为500μL，每种细胞设定复孔。培养24小时后，更换新鲜培养基并在其中一个复孔中加入诱导剂(5mmol/L丁酸钠，1μg/ml DOX和200μg/ml Cumate)设为诱导组，另外复孔仅加入等量丁酸钠设为非诱导组。继续培养48h后，以4500rpm离心15分钟方法收集病毒上清液。进行HT1080细胞Luciferase病毒滴度检测，检测方法同实施例2一致，结果如图5所示。The above-mentioned four lentivirus stable production cells were seeded in a 24-well plate with 1.5E+05 cells per well, and the culture volume was 500 μL, and each cell was set to replicate wells. After culturing for 24 hours, change the fresh medium and add inducer (5mmol/L sodium butyrate, 1μg/ml DOX and 200μg/ml Cumate) to one of the multiple wells to set the induction group, and add only the same amount of butyl to the multiple wells. Sodium is set as the non-induced group. After culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500 rpm for 15 minutes. The Luciferase virus titer of HT1080 cells was detected, and the detection method was the same as that in Example 2, and the result is shown in FIG. 5.
图5结果所示，使用TRE1-RU5(19BF124)和TRE2-RU5(19BF125)Tet嵌合响应元件调控慢病毒基因组表达盒转录的稳定生产细胞系在加入DOX和Cumate诱导下产毒滴度明显高于传统RSV-RU5和CMV-RU5嵌合启动子调控慢病毒基因组表达盒转录的稳定生产细胞系。EuLV293T-19BF124(TRE1-RU5)和EuLV293T-19BF125(TRE2-RU5)生产细胞系的诱导产毒滴度分别为3.64E+07RLU和2.37E+07RLU；分别为EuLV293T-19BF081(RSV-RU5)生产细胞的诱导产毒滴度的9.1倍和5.9倍；分别为EuLV293T-19BF123(CMV-RU5)生产细胞的诱导产毒滴度的2.46倍和1.61倍。在非诱导条件下，所有慢病毒稳定生产细胞系都检测不到病毒滴度。The results in Figure 5 show that the stable production cell line that uses TRE1-RU5 (19BF124) and TRE2-RU5 (19BF125) Tet chimeric response elements to regulate the transcription of the lentiviral genome expression cassette has significantly higher toxin production titers under the induction of DOX and Cumate A stable production cell line based on the traditional RSV-RU5 and CMV-RU5 chimeric promoters regulating the transcription of lentiviral genome expression cassettes. The induced toxin production titers of EuLV293T-19BF124 (TRE1-RU5) and EuLV293T-19BF125 (TRE2-RU5) production cell lines were 3.64E+07RLU and 2.37E+07RLU, respectively; EuLV293T-19BF081 (RSV-RU5) production cells, respectively 9.1 times and 5.9 times of the induced toxin production titers of EuLV293T-19BF123 (CMV-RU5); 2.46 times and 1.61 times of the induced toxin production titers of EuLV293T-19BF123 (CMV-RU5) production cells, respectively. Under non-induced conditions, no virus titer can be detected in all lentivirus stable production cell lines.

Claims (17)

  1. 核酸序列，其包含Tet-On系统的响应元件TRE和逆转录病毒长末端重复序列(LTR)中的R-U5功能域。Nucleic acid sequence, which includes the response element TRE of the Tet-On system and the R-U5 functional domain in the retroviral long terminal repeat (LTR).
  2. 权利要求1所述的核酸序列，其中所述R-U5功能域在TRE的TATA盒下游并与其间隔15-30bp。The nucleic acid sequence of claim 1, wherein the R-U5 functional domain is downstream of the TATA box of the TRE and separated from it by 15-30 bp.
  3. 权利要求2所述的核酸序列，其中所述R-U5功能域在TRE的TATA盒下游并与其间隔24bp。The nucleic acid sequence of claim 2, wherein the R-U5 functional domain is downstream of the TATA box of the TRE and separated from it by 24 bp.
  4. 权利要求1所述的核酸序列，其中TRE的序列如SEQ ID NO:17或SEQ ID NO:18所示。The nucleic acid sequence of claim 1, wherein the sequence of TRE is as shown in SEQ ID NO: 17 or SEQ ID NO: 18.
  5. 权利要求1所述的核酸序列，其序列如SEQ ID NO:19或SEQ ID NO:20所示。The nucleic acid sequence of claim 1, whose sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 20.
  6. 逆转录病毒基因组转录盒，其包含用于控制所述转录盒的转录的Tet-On系统的响应元件TRE或权利要求1-5中任一项的核酸序列，位于Tet-On系统的响应元件TRE或权利要求1-5中任一项的核酸序列下游的用于逆转录病毒包装的顺式作用元件以及用于插入目的核酸片段的多克隆位点。A retroviral genome transcription cassette, which comprises a Tet-On system response element TRE or the nucleic acid sequence of any one of claims 1-5 for controlling the transcription of the transcription cassette, and is located in the Tet-On system response element TRE Or a cis-acting element for retrovirus packaging downstream of the nucleic acid sequence of any one of claims 1 to 5 and a multiple cloning site for inserting the target nucleic acid fragment.
  7. 权利要求6的逆转录病毒基因组转录盒，其中所述顺式作用元件包括长末端重复序列(LTR)、引物结合位点(PBS)和病毒包装信号(phi信号)。The retroviral genome transcription cassette of claim 6, wherein the cis-acting element includes a long terminal repeat (LTR), a primer binding site (PBS), and a viral packaging signal (phi signal).
  8. 权利要求6的逆转录病毒基因组转录盒，其中所述逆转录病毒是慢病毒。The retroviral genome transcription cassette of claim 6, wherein the retrovirus is a lentivirus.
  9. 权利要求8的逆转录病毒基因组转录盒，其中所述顺式作用元件包括长末端重复序列(LTR)、引物结合位点(PBS)、病毒包装信号(phi信号)、中心多嘌呤束(cPPT)和rev蛋白应答元件(RRE)。The retroviral genome transcription cassette of claim 8, wherein the cis-acting element includes a long terminal repeat (LTR), a primer binding site (PBS), a viral packaging signal (phi signal), and a central polypurine tract (cPPT) And rev protein response element (RRE).
  10. 权利要求8的逆转录病毒基因组转录盒，其中所述顺式作用元件还包括土拨鼠肝炎病毒转录后调控序列(WPRE)。The retroviral genome transcription cassette of claim 8, wherein the cis-acting element further comprises a woodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE).
  11. 权利要求7或9的逆转录病毒基因组转录盒，其中所述长末端重复序列是能自我复制的野生型U3-R-U5序列或是删除了U3序列的自我抑制SIN序列。The retroviral genome transcription cassette of claim 7 or 9, wherein the long terminal repeat sequence is a wild-type U3-R-U5 sequence capable of self-replication or a self-suppressive SIN sequence with U3 sequence deleted.
  12. 逆转录病毒基因组转录盒，其通过在权利要求6-11中任一项的逆转录病毒基因组转录盒的多克隆位点中插入目的核酸片段获得。A retroviral genome transcription cassette, which is obtained by inserting a target nucleic acid fragment into the multiple cloning site of the retroviral genome transcription cassette of any one of claims 6-11.
  13. 载体，所述载体包含权利要求1-5中任一项的核酸序列或权利要求6-12中任一项的逆转录病毒基因组转录盒。A vector comprising the nucleic acid sequence of any one of claims 1-5 or the retroviral genome transcription cassette of any one of claims 6-12.
  14. 权利要求13的载体，其是质粒载体或病毒载体。The vector of claim 13, which is a plasmid vector or a viral vector.
  15. 宿主细胞，所述宿主细胞包含权利要求1-5中任一项的核酸序列、权利要求6-12 中任一项的逆转录病毒基因组转录盒或权利要求13或14的载体。A host cell comprising the nucleic acid sequence of any one of claims 1-5, the retroviral genome transcription cassette of any one of claims 6-12, or the vector of claim 13 or 14.
  16. 权利要求1-5中任一项的核酸序列、权利要求6-12中任一项的逆转录病毒基因组转录盒、权利要求13或14的载体、或权利要求15的宿主细胞用于生产携带目的核酸片段的逆转录病毒载体的用途。The nucleic acid sequence of any one of claims 1-5, the retroviral genome transcription cassette of any one of claims 6-12, the vector of claim 13 or 14, or the host cell of claim 15 for production and carrier purposes Use of retroviral vectors for nucleic acid fragments.
  17. 权利要求16的用途，其中权利要求1-5中任一项的核酸序列、权利要求6-12中任一项的逆转录病毒基因组转录盒、权利要求13或14的载体、或权利要求15的宿主细胞被用于所述携带目的核酸片段的逆转录病毒载体的瞬时生产或稳定生产。The use of claim 16, wherein the nucleic acid sequence of any one of claims 1-5, the retroviral genome transcription cassette of any one of claims 6-12, the vector of claim 13 or 14, or the nucleic acid sequence of claim 15 The host cell is used for transient or stable production of the retroviral vector carrying the nucleic acid fragment of interest.
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