WO2024022147A1 - Glycoprotéine de membrane baev et son utilisation - Google Patents

Glycoprotéine de membrane baev et son utilisation Download PDF

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WO2024022147A1
WO2024022147A1 PCT/CN2023/107696 CN2023107696W WO2024022147A1 WO 2024022147 A1 WO2024022147 A1 WO 2024022147A1 CN 2023107696 W CN2023107696 W CN 2023107696W WO 2024022147 A1 WO2024022147 A1 WO 2024022147A1
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envelope glycoprotein
baev
cells
seq
chimeric
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PCT/CN2023/107696
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English (en)
Chinese (zh)
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黄宇康
陈运凡
沈俊杰
徐艳敏
洪娟
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重庆精准生物技术有限公司
重庆精准生物产业技术研究院有限公司
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Priority claimed from CN202210899789.3A external-priority patent/CN117467706A/zh
Priority claimed from CN202210898270.3A external-priority patent/CN117467705A/zh
Application filed by 重庆精准生物技术有限公司, 重庆精准生物产业技术研究院有限公司 filed Critical 重庆精准生物技术有限公司
Publication of WO2024022147A1 publication Critical patent/WO2024022147A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • This application relates to lentiviruses or other retroviruses used to transduce NK, ⁇ T, ⁇ T and other cells, plasmids and cell lines used for packaging of said lentiviruses or other retroviruses, and packaging using said plasmids or cell lines Lentiviral or other retroviral methods.
  • CAR-T cell therapy has shown good results and huge potential.
  • the expression of CAR in T cells is one of the important factors affecting the efficacy of CAR-T cells.
  • CAR-T With the clinical progress of CAR-T, in order to further optimize the efficacy of CAR-T, improve the immune microenvironment and improve the persistence of CART, there are more and more solutions.
  • CAR In the design of CAR, there are higher requirements for the ability of CAR to transduce T cells.
  • NK Natural killer cells
  • ⁇ T cells for universal immune cell therapy.
  • Non-viral transduction technology represented by electroporation technology has gradually become widely recognized due to its high safety and convenient process.
  • the massive cell death caused by electroporation hinders this further promotion of technology.
  • the method of delivering mRNA through electroporation can effectively reduce toxicity and increase cell viability, this transduction is transient and is not conducive to the sustained efficacy of CAR-NK.
  • VSV-G pseudotyped lentivirus packaged with VSV-G (vesicular stomatitis virus envelope glycoprotein)
  • VSV-G vesicular stomatitis virus envelope glycoprotein
  • Patent WO2013/045639A1 announced that the modified lentivirus (BaEV lentivirus) packaged with Baboon endogenous retrovirus (Baboon endogenous virus, BaEV) envelope glycoprotein can efficiently transduce T cells and B cells.
  • BaEV envelope glycoprotein BaEV-G
  • BaEV envelope glycoprotein BaEV-G
  • BaEV-Rless which is the form of BaEV envelope glycoprotein without Fusion restrictive R peptide
  • BaEV/ TR is the form of BaEV envelope glycoprotein in which the tail domain is replaced by the tail domain of MLV envelope glycoprotein.
  • BaEV-Rless lentivirus of up to approximately 1E9TU (p24 protein measured by ELISA) can be produced in a 1L system, but the yield of this process is unstable and the titer is still difficult to meet demand (Bauler , M., et al., Production of lentiviral vectors using suspension cells grown in serum-free media. Molecular Therapy-Methods Clinical Development, 2020.17: p.58-68).
  • the BaEV/TR form Compared with BaEV-Rless, the BaEV/TR form has greatly reduced cytotoxicity and greatly reduces the appearance of syncytia during the virus packaging process, but the virus titer is lower than the BaEV-Rless form. Therefore, optimizing the structure of BaEV envelope glycoprotein and improving the packaged virus titer are the keys to whether BaEV envelope glycoprotein can be used in the production and preparation of engineered immune cells and stem cells that are difficult to transduce.
  • this application provides a packaging method for pseudoviruses, in which the envelope glycoprotein is used, and Pseudoviruses or virus particles packaged using the method described.
  • this application relates to:
  • a chimeric viral envelope glycoprotein or polypeptide includes the extracellular region, the transmembrane region of the BaEV envelope glycoprotein (BaEV-G), and the tail domain of the MoRV envelope glycoprotein.
  • the chimeric viral envelope glycoprotein or polypeptide is compared to wild-type BaEV-G only in that the chimeric viral envelope glycoprotein or polypeptide has different characteristics compared to wild-type BaEV-G.
  • the tail domain is derived from the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof.
  • the extracellular region, the transmembrane region, and the tail domain of the MoRV envelope glycoprotein of the BaEV envelope glycoprotein (BaEV-G) in the chimeric viral envelope glycoprotein or polypeptide Connect via connectors or directly.
  • the pseudovirus is a lentivirus or other retrovirus.
  • the lentivirus or other retrovirus is derived from HIV.
  • the extracellular region sequence of the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or Contains about 70% or more (such as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more) identity to the amino acid sequence shown in SEQ ID NO: 1 the sequence of.
  • the extracellular region sequence of the BaEV envelope glycoprotein is the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence of the transmembrane region of the BaEV envelope glycoprotein is the amino acid sequence shown in SEQ ID NO: 1 or 19.
  • the tail domain of the MoRV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 3 Or its functional derivative, or has about 70% or more (such as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more) identical amino acid sequences.
  • the tail domain of the MoRV envelope glycoprotein is the amino acid sequence set forth in SEQ ID NO: 3.
  • chimeric viral envelope glycoprotein or polypeptide according to item 1 which contains the amino acid sequence as in SEQ ID NO: 4 or its functional derivatives, or has more than about 70% similarity with SEQ ID NO: 4 (for example 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more) identical amino acid sequences.
  • the sequence of the chimeric viral envelope glycoprotein or polypeptide is set forth in SEQ ID NO: 4.
  • the HIV protease cleavage site is the amino acid sequence set forth in SEQ ID NO: 9.
  • the protease cleavage site sequence in the tail domain of the MoRV envelope glycoprotein that is replaced by the aforementioned amino acid sequence is the amino acid sequence shown in SEQ ID NO: 14.
  • the tail domain of the chimeric viral envelope glycoprotein or polypeptide comprises an amino acid sequence as in SEQ ID NO: 20 or a functional derivative thereof, or is about 70% identical to SEQ ID NO: 20 Amino acid sequences that are more than (eg, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) identical.
  • the tail domain of the chimeric viral envelope glycoprotein or polypeptide is the amino acid sequence in SEQ ID NO: 20.
  • the chimeric viral envelope glycoprotein or polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21, or a functional derivative thereof, or is more than 70% identical to SEQ ID NO: 21 (e.g. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more) sequence identity of the amino acid sequence.
  • the chimeric viral envelope glycoprotein or polypeptide has an amino acid sequence such as SEQ ID NO: shown in 21.
  • nucleic acid encoding the protein or polypeptide according to any one of items 1-7.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA.
  • the nucleic acid comprises both deoxyribonucleosides and ribonucleosides.
  • the nucleic acid contains chemical modifications.
  • the nucleic acid comprises a promoter for promoting expression of the protein or polypeptide.
  • the promoter is a eukaryotic promoter.
  • the promoter is selected from: CAG, miniCMV, SV40.
  • Nucleic acid according to item 8 which contains any one of the polynucleotide sequences shown in SEQ ID NO: 5, 6, 7, 8 and 27, or contains the same polynucleotide sequence as SEQ ID NO: 5, 6, 7, 8
  • the polynucleotide sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, and 27.
  • the plasmid is an envelope plasmid for viral packaging.
  • the envelope plasmid is any plasmid useful for expressing foreign proteins within eukaryotic cells.
  • the plasmid is a retroviral envelope plasmid.
  • the plasmid is a lentiviral envelope plasmid.
  • the envelope plasmid is the backbone structure of pMD2.G with the nucleic acid sequence of item 8 or item 9 inserted.
  • the plasmid is a pMD2.G plasmid in which the VSV-G nucleic acid coding sequence is replaced with the nucleic acid sequence of item 8 or item 9.
  • the envelope plasmid is a pcDNA3.1 plasmid in which the nucleic acid sequence of item 8 or item 9 is inserted.
  • the cells are selected from 293T cells, 293F cells, HEK293 cells, 293T/17SF cells.
  • the cells are immune effector cells or stem cells.
  • the cells are T cells, B cells, or NK cells.
  • composition or complex comprising:
  • the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7, according to the item 8 or 9
  • compositions or complexes are suitable for use in first, second and third generation lentiviral packaging systems.
  • the composition further comprises nucleic acids encoding Gag, Pol, Rev, and Tat, or nucleic acids encoding Gag, Pol, and Rev but not Tat.
  • the coding nucleic acids comprising Gag, Pol, Rev and Tat are present in one or two or more plasmids respectively.
  • the plasmid comprising the nucleic acid encoding Gag and Pol is pMDlg/pRRE.
  • the plasmid comprising a Rev-encoding nucleic acid is a pRSV-Rev plasmid.
  • nucleic acids encoding Gag, Pol, Rev and Tat are present in the cell according to item 11 or 12.
  • the composition further comprises nucleic acid encoding a gene sequence of interest, a promoter that drives expression of the gene of interest, an LTR, and a psi packaging signal.
  • the plasmid comprising a nucleic acid encoding a gene sequence of interest, a promoter for initiating expression of the gene of interest, an LTR, and a psi packaging signal is a transfer plasmid.
  • the present application also provides a kit for pseudovirus packaging, which includes the composition or complex in item 13.
  • the pseudovirus is a retrovirus.
  • the virus is a lentivirus.
  • the pseudovirus is a lentivirus or other retrovirus.
  • the lentivirus or other retrovirus is derived from HIV.
  • a pseudoviral particle for transducing cells which is packaged by the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7.
  • the envelope glycoprotein of the pseudovirion comprises the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7.
  • the envelope glycoprotein of the pseudovirion comprises a portion of the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7 other than the R peptide.
  • the cells are selected from: NK cells, ⁇ T cells, ⁇ T cells, DC cells, and stem cells.
  • the pseudovirus is a lentivirus or other retrovirus.
  • the lentivirus or other retrovirus is derived from HIV.
  • the The pseudoviral particles are packaged with a chimeric antigen receptor (CAR) or its coding sequence.
  • CAR chimeric antigen receptor
  • the pseudoviral particle according to item 15 further comprising the envelope glycoprotein of VSV in its envelope.
  • the protein component in the envelope consists of VSV-G and the chimeric viral envelope glycoprotein of any one of items 1-7.
  • the pseudoviral particle according to item 15 or 16 which is a pseudoviral particle of a lentivirus or other retrovirus.
  • this application relates to:
  • a method of packaging fake viruses including:
  • the cell line that stably expresses BaEV envelope glycoprotein can be a mixed clonal cell line or a screened monoclonal cell line.
  • the pseudovirus is a lentivirus or other retrovirus.
  • the lentivirus is derived from HIV.
  • nucleic acid encoding the target gene is a transfer plasmid of a lentivirus or other retroviral packaging system
  • the viral packaging element is a packaging plasmid of a lentivirus or other retroviral packaging system.
  • transposon system used for transposition is selected from the group consisting of: PB transposon system, SB transposon system, and ⁇ C31 integrase system.
  • VSV envelope glycoprotein or its coding sequence into the target cell or cell line, or the stably expressing BaEV envelope glycoprotein.
  • the cell line also stably expresses VSV envelope glycoprotein.
  • the VSV envelope glycoprotein is wild-type VSV envelope glycoprotein or a variant thereof.
  • the VSV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 18, or a functional derivative thereof, or has a sequence that is more than 70% identical to the amino acid sequence shown in SEQ ID NO: 18 Identity of the amino acid sequence.
  • the HIV protease cleavage site according to item 8 has an amino acid sequence as shown in SEQ ID NO: 9.
  • the BaEV envelope glycoprotein comprises the extracellular region, the transmembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain .
  • the BaEV envelope glycoprotein includes the extracellular region, the transmembrane region, the intracellular juxtamembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain.
  • the BaEV envelope glycoprotein includes the signal peptide, extracellular region, transmembrane region, intracellular segment juxtamembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain.
  • the BaEV envelope glycoprotein is different from wild-type BaEV-G only in that it has a different tail domain relative to wild-type BaEV-G, and the tail domain is derived from From the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof.
  • the signal peptide, extracellular region, transmembrane region, intracellular segment juxtamembrane region of the BaEV envelope glycoprotein (BaEV-G), and/or the tail domain of the MoRV envelope glycoprotein The linkage is via a linker or directly via valency (e.g. peptide bond).
  • the extracellular region sequence of the BaEV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or contains the same amino acid sequence as SEQ ID NO: 1
  • the amino acid sequences shown are sequences with more than 70% identity.
  • transmembrane region sequence of the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 2 or 19 or a functional derivative thereof, or includes the same amino acid sequence as SEQ ID NO: 2 or 19.
  • the amino acid sequence represented by NO: 2 or 19 is an amino acid sequence having at least 70% identity.
  • the MoRV viral envelope glycoprotein tail domain comprises the amino acid sequence shown in SEQ ID NO: 3 or 20 or a functional derivative thereof, or Contains an amino acid sequence having more than 70% identity with the amino acid sequence shown in SEQ ID NO: 3 or 20.
  • the amino acid sequence of the protease cleavage site in the tail domain of the MoRV viral envelope glycoprotein is as shown in SEQ ID NO: 14.
  • BaEV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 4 or a functional derivative thereof, or has the same amino acid sequence as SEQ ID NO: 4. Sequences with more than 70% identity.
  • the BaEV envelope glycoprotein encoding nucleic acid comprises a polynucleotide sequence selected from any one of SEQ ID NO: 5, 6, 7, 8, 27, or a polynucleotide sequence similar to SEQ ID NO: 5, Any one of the polynucleotide sequences in 6, 7, 8 and 27 has a polynucleotide sequence with more than 70% identity.
  • the target cell is a 293T cell or a derivative thereof.
  • the target cells are selected from the group consisting of 293T cells, 293T/17 cells, 293F cells, HEK293 cells, and 293T/17SF cells.
  • a chimeric BaEV envelope glycoprotein or polypeptide for pseudovirus packaging the tail domain of which is the BaEV envelope glycoprotein or the envelope glycoprotein tail domain of a non-BaEV envelope virus, and whose The protease cleavage site in the tail domain is replaced by the HIV protease cleavage site.
  • the amino acid sequence of the HIV protease cleavage site is shown in SEQ ID NO: 9.
  • BaEV chimeric envelope glycoprotein or polypeptide according to item 18 or 19, wherein the pseudovirus is a retrovirus.
  • the BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-21, wherein The BaEV chimeric envelope glycoprotein contains R peptide.
  • BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-22, wherein the BaEV chimeric envelope glycoprotein includes the extracellular region and the transmembrane region of the wild-type BaEV envelope glycoprotein. , and the tail domain of wild-type MoRV viral envelope glycoprotein.
  • BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-26, wherein the BaEV chimeric envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 4 or 21 or its Functional derivatives, or amino acid sequences with more than 70% identity to the amino acid sequence shown in SEQ ID NO: 4 or 21.
  • the wild-type virus of the pseudovirus is itself an enveloped virus. In some embodiments, the wild-type virus of the pseudovirus is not itself an enveloped virus. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the pseudovirus is a retroviral or lentiviral vector.
  • FIG. 1 Schematic diagram of the structure of wild-type BaEV envelope glycoprotein.
  • FIG. 1 Schematic diagram of the structure of the BaEV chimeric envelope glycoprotein with the tail replaced.
  • FIG. 1 Flow cytometric detection results of positive markers (GFP or CD19-CAR) after infection with 293T using SERV-Rless envelope glycoprotein for lentiviral packaging.
  • Flow cytometry test results of transduction efficiency (expressed as CAR-NK positive rate).
  • FIG. 9 Shows that the BaEV-MoRV tail envelope glycoprotein encoding nucleic acid was transposed into the 293T cell genome, and 293T cells stably expressing the BaEV-MoRV tail envelope glycoprotein were successfully constructed.
  • Figure 13 For difficult-to-transfect cells such as immune cells, test results of CAR delivery and transduction efficiency of enveloped lentivirus produced by different packaging methods.
  • FIG. 15 Comparison of packaging efficiency of enveloped lentivirus constructed through different methods.
  • PB means transposition method
  • LV means lentiviral transduction method.
  • FIG 16. 293T-BaEV-Rless (PB), a lentivirus packaged using a cell line constructed by PB transposon transposition (left) and 293T-BaEV-Rless (LV), a lentivirus constructed using lentiviral transposition Efficiency of transducing PBNK cells with cell line packaged lentivirus (right).
  • PB PB transposon transposition
  • LV 293T-BaEV-Rless
  • FIG. 1 Transduction efficiency of PBNK using lentivirus packaged with different target genes using the 293T-BaEV-Rless cell line.
  • Figure 20 Efficiency of transducing mouse B cells using 293T-BaEV-Rless packaged BaEV-Rless enveloped virus.
  • the present application provides a chimeric envelope glycoprotein/polypeptide, an engineered cell expressing the polypeptide, a method for packaging a pseudovirus with high transfection/transduction efficiency using the engineered cell expressing the polypeptide, and a method packaged by the method.
  • Fake virus The chimeric envelope glycoprotein/polypeptide retains the R peptide required to inhibit the toxicity of the envelope glycoprotein, reducing its toxicity, and the conditionally sheared form ensures its activity when forming a virus; the method It saves the steps of fake virus packaging and improves packaging efficiency.
  • Viruses are available for cells that are difficult to transduce, such as immune cells, stem cells, primary cells, etc. The virus can be concentrated and purified by conventional methods, and stored at -80°C for a long time.
  • transduction refers to the process by which natural or artificially engineered viral particles enter a cell and bring the genetic material contained therein into said cell.
  • envelope glycoprotein as used herein includes "wild-type” envelope glycoprotein and engineered envelope glycoprotein, such as chimeric envelope glycoprotein or wild-type envelope glycoprotein with the envelope glycoprotein removed.
  • a capsule glycoprotein formed by adding a portion of a domain or adding a portion of a domain derived from other capsule glycoproteins. But when referring to a part of the "envelope glycoprotein” of a particular virus, we are referring to that part of the "wild-type" envelope glycoprotein of that particular virus.
  • the tail domain of MoRV envelope glycoprotein refers to the tail domain of wild-type MoRV envelope glycoprotein.
  • Wild-type proteins include any reference protein mentioned herein and naturally occurring variants thereof.
  • any reference to a certain part of the envelope glycoprotein of a specific virus or a certain amino acid position thereof refers to its corresponding part or corresponding amino acid position relative to the wild-type protein.
  • this application uses one of the wild-type proteins as a reference protein.
  • the specific reference proteins listed in this application their specific sequences and the division of functional segments of the sequences can be obtained by those skilled in the art through known databases.
  • the reference protein of human endogenous viral envelope glycoprotein (HERV-G) is as shown in NCBI GeneBank No.
  • AAM68163.1, and the reference protein of koala retrovirus envelope glycoprotein (KLV-G) is as shown in NCBI GeneBank.
  • Reference number ALX81658.1, reference protein of gibbon leukemia virus envelope glycoprotein (GaLV-G) is shown as NCBI GeneBank number AAC96085.1, reference protein of murine endogenous retrovirus envelope glycoprotein (MoRV-G)
  • the reference protein is shown in NCBI GeneBank number AAC42271.1
  • the reference protein of feline leukemia virus envelope glycoprotein (FLV-G) is shown in NCBI GeneBank number ACB05740.1
  • the reference protein of feline endogenous virus envelope glycoprotein (RD114- The reference protein of G) is shown as NCBI GeneBank No.
  • the reference protein of simian endogenous retroviral envelope glycoprotein (SERV-G) is shown as NCBI GeneBank No. AEJ22866.1, and the reference protein of murine leukemia virus vesicle
  • the reference protein of membrane glycoprotein (MLV-G) is shown as NCBI GeneBank number AAP13891.1. in this application , unless otherwise stated, when referring to, for example, the tail domain of MoRV-G, it refers to the part of the MoRV-G corresponding to the tail domain of NCBI GeneBank No. AAC42271.1.
  • vector refers to a vector through which a polynucleotide sequence (eg, a gene sequence of interest) can be introduced into a host cell to transform the host and facilitate expression (eg, transcription and translation) of the introduced sequence.
  • a polynucleotide sequence eg, a gene sequence of interest
  • Vectors include plasmids, phages, viruses, artificial nanoparticles, etc.
  • artificial nanoparticle refers to artificially synthesized or artificially engineered particles with a diameter of less than 1000 nm, which are suitable for delivering the nucleic acids and/or proteins of the present application into cells.
  • exemplary artificial nanoparticles include, but are not limited to: lipid nanoparticles, exosomes, and the like.
  • the "lipid nanoparticles” are typically spherical vesicle structures composed of a single or multilamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer.
  • the nanoparticles can be made from several different types of lipids; however, phospholipids are most commonly used to generate lipid nanoparticles.
  • lipid nanoparticle formation is spontaneous when lipid films are mixed with aqueous solutions
  • formation of lipid nanoparticles can also be accelerated by applying force in the form of shaking using a homogenizer, sonicator, or extrusion device .
  • Several other additives can be added to lipid nanoparticles in order to modify their structure and properties.
  • cholesterol or sphingomyelin can be added to the lipid nanoparticle mixture to help stabilize the lipid nanoparticle structure and prevent leakage of the lipid nanoparticle inner cargo.
  • Lipid nanoparticle formulations may consist essentially of natural phospholipids and lipids such as 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin and monosialoganglioside. May be provided as solid nanoparticles (eg metals such as silver, gold, iron, titanium), non-metals, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof.
  • a "gene of interest” refers to a gene or its coding sequence contained in a vector, such as a viral vector, intended to initiate expression in a target cell by introducing the vector into the target cell.
  • lentiviral vector and “lentiviral particle” in this application can be used interchangeably, and both refer to pseudotyped lentiviral particles packaging the target gene sequence.
  • the construction method of lentiviral vectors is known in the art and is specifically described in documents such as Naldini et al. (2000) Adv.Virus.Res.55:599 609 and Negre et al. (2002) Biochimie84:1161-1171.
  • lentiviral vector particles comprise at least the following components: (i) an envelope component (“envelope” and “envelope” are used interchangeably in this application), which consist of binding to an envelope protein; consists of a phospholipid bilayer, in which the envelope
  • the protein comprises at least a chimeric or modified glycoprotein as defined above, said envelope surrounding (ii) a core component consisting of a binding of gag proteins, which itself surrounds (iii) a genome usually composed of ribonucleic acid (RNA) component and (iv) enzyme component (pol).
  • RNA ribonucleic acid
  • poly enzyme component
  • the biological material may be present within the envelope, within the core and/or within the genomic component.
  • Lentiviral vectors can be readily prepared by those skilled in the art, for example, by following the general guidance provided by Sandrin et al. (2002) Biood 100:823 832. Briefly, lentiviral vector particles can be generated by co-expressing packaging elements (i.e., core and enzyme components), genomic components, and envelope components in producer cells (eg, 293T human embryonic kidney cells or cells derived therefrom). Typically 3 to 4 plasmids can be used, but the number of plasmids can be higher depending on the extent to which the lentiviral components are broken down into individual elements.
  • packaging elements i.e., core and enzyme components
  • genomic components eg, and envelope components
  • producer cells eg, 293T human embryonic kidney cells or cells derived therefrom.
  • 3 to 4 plasmids can be used, but the number of plasmids can be higher depending on the extent to which the lentiviral components are broken down into individual elements.
  • the partial components such as envelope components, enzyme components, etc.
  • the production cell is used for packaging of viral vectors.
  • the packaging elements and envelope components may be present in plasmids, a plasmid comprising viral genome components, a plasmid comprising the envelope components, and a plasmid comprising enzyme components and/or core components.
  • the components divided into protein coding sequences are called transfer plasmids, packaging plasmids and envelope plasmids respectively.
  • lentiviral packaging plasmids include psPAX2, as well as pMDlg/pRRE and pRSV-Rev, which are components of the second- and third-generation lentiviral packaging systems, respectively.
  • the psPAX2 plasmid also contains the coding sequences for gag, pol, rev and tat
  • the pMDlg/pRRE plasmid contains the coding sequences for gag and pol
  • pRSV-Rev contains the coding sequence for rev.
  • pseudovirus and “pseudovirion” are used interchangeably and refer to viral vectors containing foreign viral envelope glycoproteins.
  • a viral vector according to the present application may be pseudotyped using a chimeric envelope glycoprotein as defined below or a variant of said envelope glycoprotein.
  • Pseudoviruses include “pseudotyped lentiviruses” and other pseudotyped retroviruses.
  • lentivirus is the collective name for "pseudotype lentivirus", wild-type lentivirus and other engineered lentivirus.
  • retrovirus is a collective term for "pseudotyped retroviruses", wild-type retroviruses and other engineered retroviruses. Those skilled in the art will know that lentivirus is a type of retrovirus.
  • viral vector is a type of “viral particle”. "Viral vector” emphasizes that the virus particles are engineered viruses that contain artificially introduced or modified proteins or nucleic acid fragments.
  • BaEV baboon endogenous retrovirus
  • BaEV-G BaEV envelope glycoprotein
  • BaEV envelope glycoprotein is specifically described in Benveniste et al. (1974) Nature 248:17-20 and Todaro et al. (1974) Cell 2:55-61.
  • the BaEV envelope glycoprotein described in the present application contains the amino acid sequence shown in SEQ ID NO: 13 sequence, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to the sequence set forth in SEQ ID NO: 13, provided that the amino acid sequence remains as determined by SEQ ID NO: 13
  • the basic function of the protein or polypeptide, the difference relative to SEQ ID NO: 13 does not result in the ability of the glycoprotein to adsorb the cell membrane of the host cell, fuse with the host cell membrane, and assist in injecting the genomic nucleic acid or the nucleic acid encoding the target gene into the host cell. loss.
  • the BaEV chimeric envelope glycoprotein is a chimeric protein form in which certain parts of the BaEV envelope glycoprotein except the extracellular region are replaced with domains of other viral envelope glycoproteins.
  • BaEV/TR contains or consists of a chimeric envelope glycoprotein consisting of a fusion of the transmembrane and extracellular regions of the BaEV envelope glycoprotein and the tail domain of the MLV (murine leukemia virus) envelope glycoprotein.
  • BaEVRLess refers to the modified BaEV envelope glycoprotein lacking the fusion inhibitory R peptide in the tail domain. The specific forms of "BaEVRLess" and “BaEV/TR” are described in detail in Chinese patent CN104080917B. .
  • fusion-inhibitory R peptide refers to the C-terminal portion of the tail domain of the envelope glycoprotein, which carries the tyrosine endocytosis signal-YXXL and matures during viral particle It is cleaved by viral protease during the process, thereby enhancing the membrane fusion ability of envelope glycoprotein.
  • the fusion-inhibitory R peptide of BaEV envelope glycoprotein is usually located between amino acid sequence 547 and 564 of the wild-type BaEV envelope glycoprotein. Therefore, when referring to a "tail domain”, unless otherwise specified, the "tail domain” includes the R peptide.
  • the envelope glycoprotein From the amino terminus to the carboxyl terminus, the envelope glycoprotein usually contains an extracellular region, a transmembrane region, an intracellular segment, a juxtamembrane region, and a cytoplasmic tail domain (Cytoplasmic tail domain, sometimes represented by "tail” in this application).
  • the transmembrane region passes through the viral envelope and is connected to the extracellular region located outside the viral envelope and the tail domain located inside the viral envelope.
  • the "extracellular region” is the part corresponding to amino acid positions 1-503 (including the endpoints) of the reference BaEV envelope glycoprotein (NCBI sequence registration number: YP_009109691.1), and the "transmembrane region” is the part corresponding to The reference BaEV envelope glycoprotein amino acid position 504 to 524 (including the endpoint) or the part of the amino acid 504 to 532 (including the endpoint), the "intracellular segment juxtamembrane region” corresponds to the reference BaEV capsule.
  • the portion of the membrane glycoprotein at amino acid positions 525 to 532 (inclusive), and the intracellular domain is the portion corresponding to amino acid positions 534 to 563 (inclusive) of the reference BaEV envelope glycoprotein.
  • the "transmembrane region” in this application may or may not include the intracellular segment juxtamembrane region.
  • “functional derivatives” of a protein include various variants or functional domains of the protein as long as the variants or functional domains retain the properties of a functional domain of the protein.
  • the function (whether it is an enhanced function or a weakened function) can be called a functional derivative of the protein.
  • chimeric antigen receptor refers to a group of engineered polypeptides or proteins that, when in immune effector cells, bind to a specific antigen contained on a target cell and upon recognition of said The specific antigen generates an intracellular signal and activates the downstream pathway of the cell where the receptor is located to initiate the killing effect of the immune effector cells on the target cells.
  • the immune effector cells include but are not limited to NK cells, macrophages, neutrophils, T cells, etc.
  • CARs generally include at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain can specifically recognize an antigen.
  • Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragmented antigen-binding regions (Fab) selected from libraries, single-domain fragments, or combinations thereof.
  • scFv single-chain variable fragments
  • Fab fragmented antigen-binding regions
  • the extracellular antigen binding region may comprise scFv, Fab or natural ligands, as well as any derivatives thereof.
  • An extracellular antigen-binding region may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and which may bind the antigen to which the intact antibody binds.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, linear antibodies; single chain antibody molecules (eg, scFv); and those formed from antibody fragments Multispecific antibodies.
  • the "signal transduction domain” usually includes immune-receptor tyrosine-based activation motifs (ITAM), whose basic composition is: YXXL/V. Among them, Y is tyrosine, L/V refers to leucine or valine, and X can be any amino acid.
  • ITAM immune-receptor tyrosine-based activation motifs
  • the tyrosine in the ITMA linked to it can be phosphorylated by PTK, a type of protein tyrosine kinase associated with the cell membrane, thereby recruiting other free protein kinases in the cell. or adapter proteins that transmit activation signals into cells.
  • the "signal transduction domain” is selected from the intracellular signal transduction domain of TCR ⁇ (CD3 ⁇ ) or Fc ⁇ RI ⁇ .
  • the "costimulatory domain” is also called a "costimulatory signal domain” and is mainly used to provide a costimulatory signal to enhance the ability of immune cells, including, for example, enhancing the proliferation, survival and/or development of memory cells.
  • the "costimulatory domain” is selected from CD28, 4-1BB (CD137), OX40 (CD134), and the like.
  • the "transmembrane domain” also known as the “transmembrane region” refers to a thermodynamically stable protein structural region anchored in the cell membrane. Transmembrane domains can be obtained from natural proteins, such as those derived from T cell receptors (TCR). In some embodiments, the transmembrane domain is selected from the group consisting of CD4, CD8 ⁇ , CD28, and CD3 ⁇ .
  • linker is a short peptide used to connect multiple domains or components in a protein or polypeptide.
  • the BaEV-MoRV-tail envelope glycoprotein in this application contains BaEV
  • the extracellular region, transmembrane region, and tail domain of envelope glycoproteins can be connected through linkers or other amino acid chains with certain functions, or directly.
  • directly connected means that the domains or components do not contain any other amino acid residues between them.
  • HEK 293T cells are an immortalized cell line derived from human embryonic kidneys.
  • HEK 293T cells are a cell line derived from HEK 293 cells through genetic technology.
  • HEK 293 cells are transfected with the adenovirus E1A gene and can stably express the SV40 large T antigen and contain the SV40 replication origin and promoter region.
  • HEK 293 cells and all other derivative cells of HEK293 cells are classified as "derivative cells of 293T cells" in this application, including but not limited to 293F cells and 293T/17SF cells.
  • protease cleavage site refers to a stretch of amino acid sequence contained in the tail domain of a retroviral envelope glycoprotein that is recognized for cleavage by the protease expressing it. When the protease recognizes the "protease cleavage site” and completes the cleavage, the tail domain of the viral envelope glycoprotein will lose the R peptide.
  • the protease cleavage sites contained in various envelope glycoproteins used in the present application are known in the art.
  • the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 14.
  • the amino acid sequence of the HIV protease cleavage site is shown in SEQ ID NO: 9.
  • a protease cleavage site of a specific envelope glycoprotein it refers to the protease cleavage site contained in the tail domain of the wild-type protein of the specific envelope glycoprotein.
  • the present application provides a chimeric viral envelope glycoprotein (also referred to as “chimeric envelope glycoprotein”) or polypeptide for pseudovirus packaging.
  • envelope glycoprotein also known as “envelope glycoprotein (Glycoprotein, GP)
  • the envelope glycoprotein is encoded by the viral genome and is coated in the outer layer of the virus.
  • GP is a multifunctional protein that plays a crucial role in virus adsorption, penetration into host cells, pathogenicity, downregulation of host cell surface protein expression, and increased virus assembly and budding. Therefore, the choice of envelope glycoproteins plays a critical role in both viral packaging titers and transduction of host cells.
  • the chimeric viral envelope glycoprotein or polypeptide provided by the present application is composed of multiple segments derived from different envelope glycoproteins, that is, the "chimeric viral envelope glycoprotein” includes at least two viral envelope glycoproteins. Domain or peptide segment of a membrane glycoprotein.
  • the chimeric viral envelope glycoprotein or polypeptide comprises the extracellular region of BaEV envelope glycoprotein (BaEV-G), the transmembrane region, and the MoRV (murine endogenous retrovirus) envelope glycoprotein The tail domain of the protein.
  • the chimerism Compared with wild-type BaEV-G, the only difference between the vesicle glycoprotein or polypeptide and the chimeric virus envelope glycoprotein or polypeptide is that the chimeric virus envelope glycoprotein or polypeptide has a different cytological tail domain than the wild-type BaEV-G, and the cytotoxicity
  • the tail domain is derived from the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof.
  • the extracellular region, the transmembrane region, and the tail domain of the MoRV envelope glycoprotein of the BaEV envelope glycoprotein (BaEV-G) in the chimeric viral envelope glycoprotein or polypeptide Connect via connectors or directly.
  • the embodiments of this application take lentivirus as an example to test the impact of the chimeric virus envelope glycoprotein on virus particle packaging and transduction, and confirm that the virus packaged using the chimeric virus envelope glycoprotein is more effective than the current
  • VSV-G or other variants of BaEV-G to package viruses with higher packaging efficiency and packaging stability.
  • chimeric viral envelope glycoprotein or polypeptide provided in this application for packaging pseudoviruses can not only be used for packaging lentiviruses or retroviruses, but can also be applied to other enveloped viruses. packaging, and have similar effects as described above on the viruses packaged by it.
  • the extracellular region sequence of the exemplary BaEV envelope glycoprotein is the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or has about 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 amino acid sequence.
  • the transmembrane region of the exemplary BaEV envelope glycoprotein is the sequence shown in SEQ ID NO: 2 or 19 or a functional derivative thereof, or an amino acid sequence having about 70% or more identity with SEQ ID NO: 2 or 19.
  • the tail domain of an exemplary MoRV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 3 or a functional derivative thereof, or an amino acid sequence having about 70% or more identity with SEQ ID NO: 3.
  • the tail domain contains an R peptide segment, and the packaging is completed by the R peptide segment.
  • the chimeric viral envelope glycoprotein may not include the R peptide segment of the tail.
  • the location and sequence of the R peptide segment of MoRV are known in the art.
  • the chimeric viral envelope glycoprotein or polypeptide is BaEV-MoRV tail envelope glycoprotein, and an exemplary amino acid sequence is the amino acid sequence of SEQ ID NO: 4 or a functional derivative thereof, or with SEQ ID NO: 4 is an amino acid sequence with about 70% or more identity.
  • the chimeric viral envelope glycoprotein or polypeptide comprises the entire sequence of wild-type BaEV-G except for the tail domain.
  • the wild-type BaEV-G and the chimeric viral envelope glycoprotein or polypeptide both comprise the amino acid sequence shown in SEQ ID NO: 13 or a functional derivative thereof, or are identical to SEQ ID NO: 13 Amino acid sequences with 70% sequence identity.
  • the term "signal peptide” refers to a short peptide chain, typically 5-30 amino acids in length, that directs the transfer of newly synthesized proteins to the secretory pathway.
  • the signal peptide is an amino acid sequence used to direct the transmembrane transfer (localization) of a protein. In most cases, the signal peptide is located at the N-terminus of the amino acid sequence. In mRNA, the coding sequence of the signal peptide is usually located after the start codon and is an RNA region encoding a hydrophobic amino acid sequence.
  • the signal peptide After the signal peptide guides the protein to complete its positioning, it is usually cleaved by the action of signal peptidase. Modification or modification of signal peptide molecules can alter or improve the transfer, localization or assembly properties of the protein, which is well known in the art. Therefore, the chimeric viral envelope glycoprotein or polypeptide provided in this application may not include a signal peptide, or the signal peptide may be replaced or modified. The signal peptide used does not need to be replaced by the exemplary signal peptide in SEQ ID NO: 11. limit.
  • the protease cleavage site of the tail domain of the MoRV envelope glycoprotein is replaced by an HIV protease cleavage site.
  • the amino acid sequence shown in SEQ ID NO: 14 is replaced by the amino acid sequence shown in SEQ ID NO: 9.
  • the tail domain of the chimeric enveloped virus glycoprotein or polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or a functional derivative thereof, or is identical to the amino acid sequence set forth in SEQ ID NO: 20 Amino acid sequences with about 70% or more identity.
  • the chimeric viral envelope glycoprotein or polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21, or a functional derivative thereof, or has 70% sequence identity with SEQ ID NO: 21 amino acid sequence. In some embodiments, the amino acid sequence of the chimeric viral envelope glycoprotein or polypeptide is set forth in SEQ ID NO: 21.
  • the present application also provides a BaEV chimeric envelope glycoprotein or polypeptide in which the protease cleavage site of the tail domain is replaced by the HIV protease cleavage site.
  • the sequence of the HIV protease cleavage site is shown in SEQ ID NO: 9.
  • the tail domain of the BaEV chimeric envelope glycoprotein or polypeptide is the tail domain of a wild-type BaEV envelope glycoprotein.
  • the tail domain of the BaEV chimeric envelope glycoprotein or polypeptide is not the tail domain of the wild-type BaEV envelope glycoprotein, but is replaced with the envelope of an envelope virus other than BaEV. Glycoprotein tail domain.
  • the tail domain of the BaEV chimeric envelope glycoprotein or polypeptide is replaced with the envelope glycoprotein tail domain of FLV, KoRV, GaLV, MoRV or MLV, and the MLV or MoRV
  • the tail domain of the envelope glycoprotein contains or does not contain R Peptides.
  • the BaEV chimeric envelope glycoprotein or polypeptide used for pseudovirus packaging is selected from: BaEV-MoRV-tail, BaEVRless, BaEV/TR, BaEV-FLV-tail, BaEV-KoRV-tail, BaEV-GaLV-tail.
  • the exemplary structures of BaEV-MoRV-tail, BaEVRless, BaEV/TR, BaEV-FLV-tail, BaEV-KoRV-tail, and BaEV-GaLV-tail are specifically described in Example 1.
  • this application also discloses a nucleic acid encoding the aforementioned chimeric virus envelope glycoprotein.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA.
  • the nucleic acid comprises both deoxyribonucleosides and ribonucleosides.
  • the nucleic acid contains chemical modifications.
  • the nucleic acid comprises a promoter for promoting expression of the protein or polypeptide.
  • the promoter is a eukaryotic promoter.
  • the promoter is selected from: CAG, miniCMV, SV40.
  • the nucleic acids are codon-optimized for different host cells.
  • the nucleic acid of the present application can be circular, linear, single-stranded or double-stranded.
  • the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 5, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Polynucleotide sequences with more than % identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 6, or is about 70% identical to any of the polynucleotide sequences in SEQ ID NO: 5, 6, 7, 8 and 27 Sequences with the above identity.
  • the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 7, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Sequences with more than % identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 5, 6 and 7, or a polynucleotide sequence corresponding to any one of SEQ ID NO: 5, 6, 7, 8 and 27 Sequences with about 70% or more identity.
  • the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 8, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Polynucleotide sequences with more than % identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 27, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Polynucleotide sequences with more than % identity.
  • the plasmid is an envelope plasmid for viral packaging.
  • the envelope plasmid is any plasmid useful for expressing foreign proteins within eukaryotic cells.
  • the plasmid is a retroviral envelope plasmid.
  • the plasmid is a lentiviral envelope plasmid.
  • the envelope plasmid is the backbone structure of pMD2.G with the nucleic acid sequence of item 8 or item 9 inserted.
  • the plasmid is a pMD2.G plasmid in which the VSV-G nucleic acid coding sequence is replaced with the nucleic acid sequence of item 8 or item 9.
  • the envelope plasmid is a pcDNA3.1 plasmid in which the nucleic acid sequence of item 8 or item 9 is inserted.
  • the viral particles are any viral particles or viral vectors that can infect or transduce the target cells of interest and express therein, introduce into the cytoplasm of the target cells, or insert the aforementioned nucleic acid into the genome of the target cells.
  • the target cells are eukaryotic cells
  • common viral particles include, but are not limited to: lentiviral vectors (LV), adenoviral vectors (ADV), adeno-associated virus vectors (AAV), Mouse leukemia virus (MLV), etc.
  • exemplary artificial nanoparticles include, for example, lipid nanoparticles, quantum dots, and the like.
  • the present application also provides engineered cells comprising the aforementioned chimeric viral envelope glycoprotein or polypeptide, or the aforementioned nucleic acid, plasmid, virus particle, or artificial nanoparticle.
  • the cells can express the aforementioned embedded cells transiently or over a period of time simply due to the introduction of the viral particles, plasmids, or artificial nanoparticles containing the previously described nucleic acids, or any of the aforementioned nucleic acids. Synthesized viral envelope glycoprotein or polypeptide.
  • the nucleic acid, plasmid, viral particle, or artificial nanoparticle further comprises a transposable element, which inserts the aforementioned nucleic acid into the engineered cell, and the engineered cell is constructed to stably express the aforementioned chimeric Cells of viral envelope glycoproteins or polypeptides.
  • composition or complex composition or complex
  • the present application also provides a composition or complex comprising the aforementioned chimeric viral envelope glycoprotein or polypeptide, the aforementioned nucleic acid, plasmid, virus particle, artificial nanoparticle, or cell.
  • the composition or complex further comprises a VSV envelope glycoprotein or a nucleic acid, plasmid, viral particle or artificial nanoparticle encoding a VSV envelope glycoprotein.
  • the composition or complex can be used for packaging of pseudoviruses, and provides the aforementioned chimeric virus envelope glycoprotein or polypeptide for packaging of pseudoviruses.
  • the compositions or complexes are suitable for use in first, second and third generation lentiviral packaging systems.
  • the composition further comprises nucleic acids encoding Gag, Pol, Rev, and Tat, or nucleic acids encoding Gag, Pol, and Rev but not Tat.
  • the coding nucleic acids comprising Gag, Pol, Rev and Tat are present in one or two plasmids respectively.
  • the plasmid comprising the nucleic acid encoding Gag and Pol is pMDlg/pRRE.
  • the plasmid comprising a Rev-encoding nucleic acid is a pRSV-Rev plasmid.
  • nucleic acids encoding Gag, Pol, Rev and Tat are present in the aforementioned cells.
  • the composition further comprises nucleic acid encoding a gene sequence of interest, a promoter that drives expression of the gene of interest, an LTR, and a psi packaging signal.
  • the nucleic acid comprising a sequence encoding a gene of interest, a promoter for initiating expression of the gene of interest, an LTR and a psi packaging signal is a transfer plasmid.
  • the composition or complex includes a cell with a nucleic acid capable of stably expressing the aforementioned chimeric viral envelope glycoprotein or polypeptide inserted into the genome, which can be used to package the gene sequence of interest into pseudoviral particles. Therefore, the composition or complex further includes nucleic acid encoding a gene sequence of interest, a promoter for initiating expression of the gene of interest, LTR and psi packaging signals, and other auxiliary components for virus packaging. For example, in some embodiments, the composition or complex further comprises a nucleic acid encoding Gag, Pol, Rev and/or Tat.
  • the nucleic acid encoding the target gene sequence, the promoter for initiating the expression of the target gene, the LTR and the psi packaging signal, and the coding nucleic acid containing Gag, Pol, Rev and/or Tat can be plasmids or genome components that are convenient for use in the cells. in the form of expression or packaging.
  • the nucleic acid expressing the aforementioned chimeric viral envelope glycoprotein or polypeptide is inserted into the genome of the cell through viral transduction or transposition.
  • the complex or composition includes the aforementioned chimeric viral envelope glycoprotein or polypeptide, which may be complexed or complexed with other nucleic acids or proteins that package the required elements.
  • the complex may be a nucleoprotein formed by the complex of protein and nucleic acid.
  • the present application also provides a kit for pseudovirus packaging, which includes the aforementioned composition or complex.
  • the pseudovirus is a retrovirus.
  • the virus is a lentivirus.
  • the present application also provides a pseudoviral particle for transducing target cells, which contains the aforementioned chimeric viral envelope glycoprotein or polypeptide in the envelope.
  • the target cells are immune effector cells or hematopoietic stem/progenitor cells.
  • the target cells are selected from: NK cells, ⁇ T cells, ⁇ T cells, DC cells, and stem cells.
  • the pseudovirus is a lentivirus or other retrovirus.
  • the lentivirus or other Retroviruses originate from HIV.
  • the pseudoviral particle is packaged with a chimeric antigen receptor (CAR) or its coding sequence.
  • CAR chimeric antigen receptor
  • the immune effector cells after transduction using the viral vector, are modified into engineered immune effector cells that express foreign proteins, such as CAR-T cells, CAR-NK cells, etc.
  • the pseudoviral particles comprise a chimeric antigen receptor or a component thereof, or a coding sequence for the chimeric antigen receptor or a component thereof.
  • the chimeric antigen receptor specifically binds CD19 or CD123.
  • the envelope of the pseudoviral particle further contains the envelope glycoprotein of VSV.
  • the protein component in the envelope consists of VSV-G and the aforementioned chimeric viral envelope glycoprotein.
  • the VSV-G comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having more than 70% identity to the amino acid sequence set forth in SEQ ID NO: 18.
  • the present application also provides virus particles or pseudovirions in which the target gene itself contains a nucleic acid sequence encoding the aforementioned chimeric virus envelope glycoprotein or polypeptide.
  • the viral particles are selected from engineered viral vectors such as lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, and the like.
  • the envelope glycoprotein of the pseudoviral particle may or may not include the aforementioned chimeric viral envelope glycoprotein or polypeptide.
  • the target cells of the pseudoviral particles whose gene of interest itself contains the nucleic acid sequence encoding the aforementioned chimeric virus envelope glycoprotein or polypeptide are 293T cells or cells derived from them, for example, selected from: 293T/17, 293F , HEK293, 293T/17SF target cells.
  • the pseudoviral particles are lentiviral or other retroviral pseudoviral particles.
  • the lentivirus or other retrovirus is derived from the HIV virus.
  • the present application also provides cells that express or contain the aforementioned chimeric viral envelope glycoproteins or polypeptides.
  • the cells can be used for packaging of enveloped lentiviruses and provide envelope glycoproteins for the packaging.
  • the cells are 293T cells or derivatives thereof.
  • the cells are selected from 293T cells, 293F cells, HEK293 cells, 293T/17SF cells.
  • the present application also provides engineered cells transduced by viral vectors packaged by the aforementioned chimeric viral envelope glycoprotein or polypeptide.
  • the engineered cells express exogenous genes of interest, or overexpress certain endogenous genes, or regulate the expression of endogenous genes of the cells through the expression of the genes of interest, or modify the cells.
  • the target gene sequence include, but are not limited to: chimeric Antigen receptor (CAR) coding sequence, Ig gene coding sequence, cytokine gene coding sequence, shRNA, CRISPR gene editing system and other gene editing systems.
  • the cells are immune effector cells or stem cells.
  • the cell is a T cell, B cell, NK cell, DC cell, ⁇ T cell, or ⁇ T cell, for example.
  • the cells are transduced by the viral vector to form, for example, CAR-NK cells or CAR-T cells.
  • the present application also provides the use of the aforementioned chimeric virus envelope glycoprotein or polypeptide, the aforementioned nucleic acid, the aforementioned plasmid, virus particles, artificial nanoparticles, and the aforementioned cells, especially for packaging pseudoviruses.
  • the viral packaging is a lentiviral packaging or other retroviral packaging.
  • the lentivirus or other retrovirus is derived from HIV.
  • the aforementioned chimeric viral envelope glycoprotein or polypeptide includes a nucleic acid, plasmid, virus particle, or artificial nanoparticle that contains the encoding sequence of the aforementioned chimeric viral envelope glycoprotein or polypeptide and a nucleic acid encoding a gene of interest, and other elements required for virus packaging or their expression vectors, such as plasmids, together constitute a packaging system.
  • the packaging system is introduced into the target cells and uses relevant organelles, functional proteins, etc. in the target cells to package the pseudovirus.
  • the nucleic acid, plasmid, virus particle, or artificial nanoparticle containing the aforementioned chimeric viral envelope glycoprotein or polypeptide coding sequence is first introduced into the target cell, and the chimeric viral envelope glycoprotein or polypeptide is performed. Transient expression of polypeptides, or insertion into the target cell genome to construct cells that stably express chimeric viral envelope glycoproteins or polypeptides; subsequently, other nucleic acids encoding the target genes, as well as other elements required for virus packaging or their expression vectors are introduced into the cells, Or similarly, it can be inserted into the cell genome, and then the pseudovirus can be packaged using relevant organelles, functional proteins, etc. in the target cell.
  • Exemplary specific usage methods include but are not limited to the following steps:
  • the BaEV-MoRV tail is integrated into the genome of the cell line used for virus packaging using the aforementioned lentiviral vector or non-viral vector (including but not limited to artificial nanoparticles and plasmids) inserted with the BaEV-MoRV tail coding sequence.
  • the integration methods include but are not limited to lentiviral system, PB transposon system, SB transposon system, ⁇ C31 integrase system, etc.
  • the lentivirus can be selected from the aforementioned lentiviral vectors, and the PB transposon system can be selected from the aforementioned plasmids.
  • the promoter driving BaEV expression can be a promoter of different strengths, including CAG, miniCMV, SV40, etc.
  • WPRE or bGH poly A can be added to the 3' end of the ORF to improve the stability of the transcript. Qualitative. Resistance genes including but not limited to puromycin, neomycin, etc. are also added to the plasmid to facilitate the subsequent screening of cell lines.
  • the introduction can introduce the coding sequence of BaEV-MoRV-tail into the target cell genome under the action of sensitizing reagents including DEAE, polybrene, etc.
  • the target cells are selected from 293T cells or cells derived therefrom.
  • the introduction method includes but is not limited to electroporation, lipofection, calcium transfer, PEI and other methods, and the plasmid containing the transposon and transposase is introduced into the target cell.
  • different forms of BaEV coding sequences are inserted into the genome of target cells under the action of transposase.
  • cells expressing BaEV-MoRV tail can be sorted out through cell line screening methods (including but not limited to flow sorting, drug screening, etc.). On this basis, in order to further optimize the efficiency of virus packaging, the above cells can be monocloned through methods such as flow sorting and limiting dilution. It has been identified that, in some embodiments, BaEV expression abundance among different clones of the 293T-BaEV cell line is different, and clones with higher expression abundance are more advantageous in virus packaging efficiency.
  • the above-mentioned 293T cell line stably expressing BaEV-MoRV-Tail was transduced with the plasmid containing the target gene, RRE, and Rev at a certain ratio, and then the virus was packaged.
  • the culture supernatant was harvested 48 hours after transfection, and the virus was concentrated by PEG6000 for flow cytometry.
  • the titer can reach above 1e8TU/ml.
  • VSV-G encoding plasmid can be added during the packaging process to further increase the lentivirus titer by up to 5-8 times.
  • PBNK PBMC-derived NK cells
  • the present application also provides the use of the aforementioned chimeric virus envelope glycoprotein or polypeptide, the aforementioned nucleic acid, the aforementioned plasmid, viral particles, artificial nanoparticles, the aforementioned cells, the aforementioned compositions or complexes, and the aforementioned pseudoviral particles in the preparation of cells.
  • Use in therapeutic medicines include, for example, using the lentivirus packaged with the chimeric viral envelope glycoprotein or polypeptide to transduce immune effector cells to prepare engineered immune effector cells with precise targeting, such as CAR-T cells, CAR-NK cells, etc.; prepare pseudoviral particles that express the aforementioned chimeric virus envelope glycoprotein or polypeptide in the envelope.
  • the pseudoviral particles contain the target gene and can carry the target gene for transduction in vivo or in vitro. from subjects To make the cells express therapeutic exogenous proteins, to regulate the expression of endogenous genes of the cells, or to modify the cells, examples of the target gene sequences include but are not limited to: embedded Synthetic antigen receptor (CAR) coding sequence, Ig gene coding sequence, cytokine gene coding sequence, shRNA, CRISPR gene editing system and other gene editing system components.
  • CAR embedded Synthetic antigen receptor
  • the seventh aspect of the present application provides a packaging method for a pseudovirus with improved packaging efficiency.
  • the pseudovirus has high transduction efficiency for target cells that are difficult to transduce, especially immune cells.
  • target cell refers to a cell into which exogenous nucleic acid or protein is introduced for protein expression or viral packaging.
  • the packaging method of the pseudovirus includes: introducing the aforementioned BaEV envelope glycoprotein or a vector containing the aforementioned BaEV envelope glycoprotein encoding nucleic acid, the nucleic acid encoding the target gene and the viral packaging element into the target cell; or constructing a stable expression of the BaEV envelope glycoprotein. Protein-producing cell lines, and the nucleic acid encoding the target gene and viral packaging elements are introduced into the cell lines.
  • the vector may be selected from: plasmids, phages, viruses, artificial nanoparticles, etc.
  • the vector in addition to the nucleic acid encoding the BaEV envelope glycoprotein, the vector further includes a promoter that can initiate expression of the BaEV envelope glycoprotein.
  • virus packaging elements refer to regulatory elements, structural proteins (other than envelope glycoproteins) and related enzymes required for virus packaging, or nucleic acids encoding the regulatory elements, structural proteins and related enzymes.
  • virus packaging components are recorded in relevant published academic documents in the field, which can be obtained by those skilled in the art.
  • the pseudovirus is a lentivirus or other retrovirus.
  • the BaEV envelope glycoprotein or a vector comprising a BaEV envelope glycoprotein encoding nucleic acid, a target gene encoding nucleic acid and a viral packaging element, Or a cell line stably expressing BaEV envelope glycoprotein, nucleic acid encoding the target gene and viral packaging components form a lentivirus or other retrovirus packaging system.
  • the lentivirus or other retrovirus packaging system can be selected from the group consisting of first-generation, second-generation, and third-generation lentivirus packaging systems.
  • the nucleic acid encoding the gene of interest is a transfer plasmid of a lentivirus or other retroviral packaging system, which contains the LTRs and psi packaging signal of the lentivirus or other retrovirus.
  • the nucleic acid encoding the gene of interest can also refer to any nucleic acid or vector containing the nucleic acid encoding the gene of interest and LTRs and psi packaging signals of lentivirus or other retrovirus, for example, it can be a linear nucleic acid, Viral vectors, artificial nanoparticles, etc.
  • the pseudovirus is a lentivirus or other retrovirus, the packaging element of which includes the coding sequences of Gag, Pol, Rev and Tat genes.
  • the pseudovirus is slow Viruses or other retroviruses, the packaging element only contains the coding sequences of Gag, Pol and Rev genes, and the nucleic acid encoding the target gene also contains a specialized promoter.
  • the pseudovirus is a lentivirus and the "viral packaging element" is selected from the psPAX2 plasmid, or a combination of pMDlg/pRRE and pRSV-Rev plasmids.
  • psPAX2, pMDlg/pRRE and pRSV-Rev are common plasmids in the art, and the necessary functional elements are known in the art.
  • the psPAX2 plasmid contains the coding sequences of gag, pol, rev and tat
  • the pMDlg/pRRE plasmid contains the coding sequences of gag and pol
  • pRSV-Rev contains the coding sequence of rev.
  • the method described in this application is applicable to a variety of target genes, including but not limited to chimeric antigen receptors (such as CAR targeting CD19, CD123) and genes of various cytokines.
  • the pseudovirus is a lentivirus or other retrovirus
  • the lentivirus or other retrovirus is selected from: Rous sarcoma virus, Rous-related virus, chicken tumor virus, avian leukosis virus ( ALV), murine sarcoma virus (MSV), murine leukemia virus (MLV), murine endogenous viruses, pork tumour virus, bovine leukemia virus, porcine leukemia virus, murine mammary tumor virus, primate sarcoma virus, simian leukemia virus, Baboon tumor virus type C, Mason-Pfizer monkey virus (MPMV), human T-cell virus types I, II, V (HTLV-I, II, V), HIV (human immunodeficiency virus), ovine demyelinating Leukoencephalitis virus, sheep lung adenoma virus, equine infectious anemia virus (EIAV), primate foamy virus, feline foamy virus, bovine foamy virus, human foamy virus, etc.
  • ALV avian le
  • nucleic acid may refer to any form of nucleic acid, including, but not limited to, linear nucleic acid, circular nucleic acid (e.g., plasmid), genomic nucleic acid, artificially modified nucleic acid, DNA, RNA, or a nucleic acid composed of DNA and RNA. nucleic acids.
  • the nucleic acid encoding the target gene described in this application is a transfer plasmid of a lentivirus or other retroviral packaging system.
  • any method for constructing a cell line that stably expresses a foreign protein can be used to construct a cell line that stably expresses the BaEV envelope glycoprotein, for example, using simple homologous recombination.
  • constructing a cell line stably expressing the BaEV envelope glycoprotein is achieved by inserting the nucleic acid encoding the BaEV envelope glycoprotein into the genome of the target cell through lentiviral transduction or transposition.
  • the lentivirus can be lentivirus packaged using the method provided in this application or can be lentivirus packaged using traditional methods.
  • the transposition can be performed using any common transposon system, for example, the transposon system is selected from: PB transposon system, SB transposon system, ⁇ C31 integrase system.
  • the method for constructing a cell line that stably expresses foreign proteins is a gene editing method, such as CRISPR gene editing method, ZFN gene editing method, TALEN gene editing method, Mega nuclease gene editing method, etc.
  • the method for constructing a cell line stably expressing a foreign protein is a method of lentiviral transduction, which includes contacting a lentivirus comprising the BaEV envelope glycoprotein encoding nucleic acid with a target cell, or Before or after contact with the encapsulated cells, add a sensitizing reagent (or transfer-assisting reagent) DEAE or polybrene, or a reagent with the same active ingredient as DEAE or polybrene.
  • a sensitizing reagent or transfer-assisting reagent
  • DEAE or polybrene a reagent with the same active ingredient as DEAE or polybrene.
  • the active ingredients of DEAE and polybrene reagents are known in the art, and there is no difference in the active ingredients of the reagents provided by various manufacturers, and the reagents from different manufacturers will not cause significant differences in viral transduction efficiency.
  • the packaging method of the pseudovirus further includes introducing VSV envelope glycoprotein or its coding sequence into the target cell or cell line, or making the cell line stably expressing the BaEV envelope glycoprotein simultaneously VSV envelope glycoprotein is also stably expressed.
  • the VSV envelope glycoprotein is wild-type VSV envelope glycoprotein or a variant thereof.
  • the VSV envelope glycoprotein has the amino acid sequence set forth in SEQ ID NO: 18, or a functional derivative thereof, or has at least 70% sequence identity with the sequence set forth in SEQ ID NO: 18 amino acid sequence.
  • the amino acid sequence of the VSV envelope glycoprotein is set forth in SEQ ID NO: 18.
  • the BaEV envelope glycoprotein used in the present methods is a chimeric envelope glycoprotein in which the protease cleavage site in the tail domain of the envelope glycoprotein is determined by the HIV protease cleavage site. replace.
  • the amino acid sequence of the HIV protease cleavage site is as shown in SEQ ID NO: 9.
  • the aforementioned BaEV envelope glycoprotein may be a wild-type BaEV envelope glycoprotein or a modified BaEV envelope glycoprotein.
  • the modified BaEV envelope glycoprotein includes a chimeric protein whose tail domain is replaced with the tail domain of a non-BaEV envelope glycoprotein.
  • the "non-BaEV envelope glycoprotein" may refer to any other envelope glycoprotein except wild-type BaEV envelope glycoprotein, including but not limited to: FLV, KoRV, GaLV, MoRV and MLV.
  • the BaEV envelope glycoprotein comprises the extracellular region, the transmembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain.
  • the BaEV envelope glycoprotein includes the extracellular region, the transmembrane region, the intracellular juxtamembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain.
  • the BaEV envelope glycoprotein includes a signal peptide, an extracellular region, a transmembrane region, an intracellular segment and a juxtamembrane region of the BaEV envelope glycoprotein. and the tail domain of MoRV viral envelope glycoprotein.
  • the BaEV envelope glycoprotein is different from wild-type BaEV-G only in that it has a different tail domain relative to wild-type BaEV-G, and the tail domain is derived from From the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof.
  • the signal peptide, extracellular region, transmembrane region, intracellular juxtamembrane region of the BaEV envelope glycoprotein (BaEV-G), and/or the tail domain of the MoRV envelope glycoprotein Connect via connectors or directly.
  • the BaEV envelope glycoprotein is BaEV-MoRV-tail, that is, a BaEV envelope glycoprotein in which the tail domain is replaced with the tail domain of the MoRV envelope glycoprotein.
  • the BaEV envelope glycoprotein is BaEVRless, that is, the BaEV envelope glycoprotein with the R peptide in the tail domain removed.
  • the BaEV envelope glycoprotein is BaEV/TR, that is, a BaEV envelope glycoprotein in which the tail domain is replaced with the tail domain of the MLV envelope glycoprotein.
  • the extracellular region sequence of the BaEV envelope glycoprotein comprises the sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the extracellular region sequence of the BaEV envelope glycoprotein is shown in SEQ ID NO: 1. In some embodiments, the transmembrane region sequence of the BaEV envelope glycoprotein comprises a sequence as shown in SEQ ID NO: 2 or 19 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the sequence of the transmembrane region of the BaEV envelope glycoprotein is as shown in SEQ ID NO: 2 or 19.
  • the MoRV viral envelope glycoprotein tail domain comprises the sequence shown in SEQ ID NO: 3 or 20 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the MoRV viral envelope glycoprotein tail domain sequence is shown in SEQ ID NO: 3 or 20. In some embodiments, the MoRV viral envelope glycoprotein comprises SEQ ID NO: 1-3, or SEQ ID NO: 1, 3, 19, or SEQ ID NO: 1, 2, 20, Or the sequences shown in SEQ ID NO: 1, 19, 20 or functional derivatives thereof, or sequences having more than 70% identity with them.
  • the MoRV viral envelope glycoprotein sequence consists of SEQ ID NO: 1-3, or consists of SEQ ID NO: 1, 3, 19, or consists of SEQ ID NO: 1, 2, 20, or consists of The sequences of SEQ ID NO: 1, 19, and 20, or the sequences of SEQ ID NO: 1, 3, and 19 are sequentially connected.
  • the order of the following domains in the BaEV envelope glycoprotein from N-terminus to C-terminus is: BaEV-G extracellular region, BaEV-G extracellular region transmembrane region, and MoRV virus vesicle Membrane glycoprotein tail domain.
  • the extracellular region, transmembrane region, and MoRV viral envelope glycoprotein tail domain of the BaEV envelope glycoprotein are directly connected or connected through a linker.
  • the BaEV envelope glycoprotein comprises SEQ ID NO: The sequence shown in 4 or its functional derivative, or a sequence having more than 70% identity with it.
  • the BaEV envelope glycoprotein sequence is set forth in SEQ ID NO: 4.
  • the protease cleavage site shown in SEQ ID NO: 14 in the MoRV viral envelope glycoprotein tail domain is replaced with an HIV protease cleavage site.
  • the MoRV viral envelope glycoprotein comprises the sequence shown in SEQ ID NO: 21 or a functional derivative thereof, or a sequence having more than 70% identity thereto.
  • the BaEV envelope glycoprotein sequence is set forth in SEQ ID NO: 21.
  • the BaEV envelope glycoprotein-encoding nucleic acid is codon-optimized for different target cells.
  • the BaEV envelope glycoprotein encoding nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 5, 6, 7, 8, 27, or is identical to SEQ ID NO: 5, 6 , 7, 8 and 27 any one of the nucleotide sequences has more than 70% identity.
  • the promoter used to initiate the expression of BaEV envelope glycoprotein can be any promoter suitable for target cells, preferably a promoter that facilitates expression in target cells. Preferred promoters for specific target cells are known in the art.
  • the target cell is a 293T cell or a derivative cell thereof
  • the promoter of the BaEV envelope glycoprotein encoding nucleic acid is CAG, miniCMV, or SV40.
  • 293T cells or derivative cells thereof include, but are not limited to: 293T cells, 293T/17 cells, 293F cells, HEK293 cells, and 293T/17SF cells.
  • Methods to integrate BaEV of different structures into the genome of cell lines used for virus packaging include but are not limited to lentiviral systems, PB transposon systems, SB transposon systems, ⁇ C31 integrase systems, etc. Specifically, lentiviral systems and PB transposon system.
  • BaEV of different structures including BaEV-Rless, BaEV-MoRV, BaEV-HIV cleavage site (that is, BaEV-G that replaces the BaEV protease cleavage site with the HIV protease cleavage site)
  • the promoter driving BaEV expression can be promoters of different strengths, including CAG, miniCMV, SV40, etc.
  • WPRE or bGH poly A can be added to the 3' end of the ORF to improve the stability of the transcript. Resistance genes including but not limited to puromycin, neomycin, etc. are also added to the plasmid to facilitate the subsequent screening of cell lines.
  • the lentiviral system can insert the BaEV-MoRV coding sequence into the 293T genome under the action of sensitizing reagents including DEAE, polybrene, etc.
  • sensitizing reagents including DEAE, polybrene, etc.
  • the transposon and transposase plasmids need to be introduced into 293T through methods including but not limited to electroporation, lipofection, calcium transfection, PEI, etc., and different forms will be transformed under the action of transposase.
  • the BaEV coding sequence was inserted into the 293T genome.
  • 293T can be screened through cell line screening methods including but not limited to flow cytometry sorting, drug screening, etc.
  • the above cells can be monocloned through methods such as flow sorting and limiting dilution. It was identified that the BaEV expression abundance of different clones of the 293T-BaEV cell line was different, and clones with medium expression abundance had more advantages in virus packaging efficiency.
  • the transfer plasmid (such as a plasmid encoding a chimeric antigen receptor), pMDLg/pRRE and pRSV-Rev are transfected into the above-mentioned 293T-BaEV cell line at a certain ratio and then the virus is packaged.
  • the culture and supernatant are harvested 48 hours after transfection.
  • the viral flow titer can reach over 1e8TU/ml.
  • VSV-G encoding plasmid can be added during the packaging process to further increase the lentivirus titer by up to 5-8 times.
  • PBMC-derived NK PBNK
  • Cationic polymers such as polybrene and DEAE can be added before adding viruses to improve transduction efficiency.
  • the positivity rate was detected by flow cytometry, and the results showed that the positivity rate could reach 50-80%.
  • the present application also provides a pseudovirus packaged using the aforementioned packaging method, and the pseudovirus envelope contains the aforementioned chimeric envelope glycoprotein or polypeptide, and/or VSV envelope glycoprotein.
  • the wild-type virus of the pseudovirus is itself an enveloped virus. In some embodiments, the wild-type virus of the pseudovirus is not itself an enveloped virus.
  • the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the pseudovirus is a retroviral or lentiviral vector.
  • the BaEV-MoRV-tail structure can be used without missing the R peptide.
  • Adding VSV-G during the lentivirus packaging process can form BaEV::VSV-G chimeric envelope lentivirus, further improving the titer of the lentivirus and expanding its scope of application;
  • the constructed 293T-BaEV cell line can improve the transduction efficiency of the virus.
  • AAM68163.1 human endogenous viral envelope glycoprotein, HERV-G
  • ALX81658.1 koala Retroviral envelope glycoprotein, KLV-G
  • AAC96085.1 Gabbon leukemia virus envelope glycoprotein, GaLV-G
  • AAC42271.1 Murine endogenous retroviral envelope glycoprotein, MoRV-G
  • ACB05740.1 feline leukemia virus envelope glycoprotein, FLV-G
  • CAA61093.1 feline endogenous viral envelope glycoprotein, RD114-G
  • AEJ22866.1 simian endogenous retroviral envelope glycoprotein protein, SERV-G
  • AAP13891.1 murine leukemia virus envelope glycoprotein, MLV-G
  • BaEV-G BaEV envelope glycoprotein
  • SERV-Rless SEm-G with R peptide removed
  • HERV-wt wild type HERV-G
  • HERV-Rless R peptide removed
  • BaEV-FLV tail BaEV-KLV tail
  • BaEV-GaVL tail BaEV-MoRV tail coding sequences were connected to pMD2 through a series of processes such as codon optimization, sequence synthesis, double enzyme digestion, ligation, and sequencing verification.
  • the lentiviral transfer plasmid a lentiviral transfer plasmid containing a CAR (chimeric antigen receptor) coding sequence (the CAR targeting CD19 is used in this example, and its specific information is detailed in Chinese patent: CN107226867A, whose full text incorporated herein by reference) or pBKL2-GFP (a homemade plasmid, a lentiviral transfer plasmid with inserted GFP green fluorescent protein coding sequence), as well as pMDLg/pRRE and pRSV-Rev; respectively with SERV-Rless-pcDNA 3.1, HERV-wt -pcDNA 3.1, HERV-Rless-pcDNA 3.1, BaEV-FLV tail-pcDNA 3.1, BaEV-KLV tail-pcDNA 3.1, BaEV-GaVL tail-pcDNA 3.1 or BaEV-MoRV tail-pcDNA 3.1 combination, co-transfected 293T cells, Perform lentivirus packaging and
  • the concentrated lentivirus was titer tested using 293T. Inoculate 1 ⁇ 10 5 293T cells into a 24-well plate, dilute the concentrated virus 10 times, and infect 293T cells at 1, 2, and 5 ul/well respectively, and add DEAE transfer agent (Shanghai Sangon, Cat. No. : A600147), 2 days after infection, the infected 293T cells were collected for flow cytometric detection. Among them, the CAR structure positive rate (the proportion of cells containing the CAR structure in the total number of cells, hereinafter referred to as the "positive rate”) was detected by flow cytometry using PE-coupled L protein (Sino biological, product number: 11044-H07E-P).
  • the results show that the combination containing SERV-Rless cannot form lentivirus ( Figure 3), while the combination containing HERV-wt, HERV-Rless, BaEV-FLV tail, BaEV-KLV tail, BaEV-GaVL tail, and BaEV-MoRV tail can form Lentivirus ( Figure 4).
  • PBNK peripheral blood NK cells
  • Flow cytometry was performed 3 days after transduction. The results showed that the transduction efficiency was less than 5. % ( Figure 5), that is, effective transduction cannot be performed.
  • the lentivirus packaged with BaEV-MoRV tail has a relatively higher packaging titer and has a relatively higher transduction efficiency for peripheral blood NK cells.
  • BaEV-MoRV-tail envelope glycoprotein shows more prominent advantages in transduction efficiency.
  • the codon-optimized BaEV-MoRV tail coding sequence was ligated into the PB transposon plasmid (synthesized by Genscript, which contains the necessary functional components of the PiggyBac transposon) through double enzyme digestion. After verification by enzyme digestion and sequencing , amplify and extract the corresponding plasmid pPBK-CAG-BaEVRless-WPRE-bGH, and the plasmid concentration used should not be less than 1000ng/ul.
  • the electroporated 293T cells were expanded and cultured, and positive cells were sorted by flow cytometry.
  • the labeled antibody used was a mouse polyclonal antibody for the extracellular region of BaEV (homemade, which can be obtained by any polyclonal antibody known in the art).
  • the fluorescent secondary antibody is rabbit anti-mouse Alexa 647 (Thermos product number: A21239).
  • the sorted 293T cells expressing BaEV-MoRV tail (293T-BaEV-MoRV tail) were expanded and cultured and the positive rate was retested again (Figure 9). As can be seen from the figure below, the sorted 293T cells expressing BaEV-MoRV tail were expanded and cultured.
  • the 293T cells of MoRV tail still have a high positive rate of expression.
  • the above-sorted 293T cells expressing BaEV-MoRV tail (293T-BaEV-MoRV tail cell line) were transduced using the composition shown in Table 1, and the titer of the envelope lentivirus was detected.
  • the titer test results are also shown in Table 1. It can be seen that adding VSV-G and BaEV-MoRV-tail envelope glycoprotein at the same time during the packaging process can further improve the quality of lentivirus compared with only adding BaEV-MoRV-tail envelope glycoprotein. Packaging efficiency.
  • Figure 10 It can be seen that lentivirus packaged with BaEV-MoRV-tail envelope glycoprotein, whether or not it further contains VSV-G, can achieve high infection efficiency on PBNK cells.
  • Example 2 Same as Example 1; after harvesting the lentivirus crude extract, digest the 293T cells with trypsin and collect them into a centrifuge tube. After labeling 7-AAD, use a flow cytometer to detect the cell viability, as shown in the table below, by As can be seen in Table 2, the packaged lentivirus can maintain a high cell viability rate.
  • Example 4 The 293T-BaEV-MoRV Tail cell line constructed by the lentiviral method is used for lentiviral packaging of CD123-CAR and mbIL15.
  • the BaEV-MoRV Tail coding sequence was obtained through codon optimization and sequence synthesis, and was ligated into the lentiviral transfer plasmid pBKL2 using different promoters through enzyme digestion. After enzyme digestion and sequencing verification, the corresponding plasmids pBKL2-MoRV tail (the expression of envelope glycoprotein in this plasmid uses the CAG promoter), pBKL2-miniCMV-MoRV tail and pBKL2-SV40-MoRV tail were extracted.
  • pBKL2-BaEV-MoRV tail or pBKL2-miniCMV-BaEV-MoRV tail or pBKL2-SV40-BaEV-MoRV tail were transfected into 293T cells with lentiviral packaging plasmids pMDLg/pRRE and pRSV-Rev respectively through calcium transfection.
  • the virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was determined by RT-PCR.
  • transduce 293T with the above lentivirus 3 days after transduction, the transduction efficiency is detected by flow cytometry. If the positive rate is >85%, subsequent verification can be carried out.
  • the BaEV cell line positive rate detection antibody is a mouse polyclonal antibody of the extracellular region of BaEV, and the fluorescent secondary antibody is rabbit anti-mouse Alexa 647.
  • the results show that using lentivirus transduction cells can also successfully construct a cell line expressing BaEV-MoRV tail envelope glycoprotein (293T-BaEV-MoRV Tail (LV)) and be used for subsequent lentivirus packaging.
  • the lentiviral plasmids of CD123-CAR (derived from patent ZL201810207761.2) and mbIL15 and three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G were transduced into 293T expressing BaEV-MoRV Tail obtained in the above steps. cell.
  • the virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was detected by flow cytometry. Viruses can be stored at -80°C, as shown in the table 3 It can be seen that lentivirus packaging using CD123-CAR and mbIL15 as target genes can also be successfully carried out using the envelope glycoprotein of the present application.
  • the detection method please refer to Example 2. The results are shown in Figure 12. It can be seen that lentiviruses containing CD123-CAR, mbIL15 and other foreign protein coding sequences packaged with the envelope glycoprotein of the present application can also infect PBNK cells with high efficiency.
  • the BaEV-Rless coding sequence was obtained through codon optimization and sequence synthesis, and was ligated into the lentiviral transfer plasmid pBKL2 with different promoters through enzyme digestion. After restriction enzyme digestion and sequencing verification, the corresponding plasmids pBKL2-BaEVRless (CAG promoter), pBKL2-miniCMV-BaEVRless (miniCMV promoter) and pBKL2-SV40-BaEVRless (SV40 promoter) were extracted.
  • pBKL2-BaEVRless or pBKL2-miniCMV-BaEVRless or pBKL2-SV40-BaEVRless and three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G were transfected into 293T cells by calcium transfection method.
  • the virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was determined by RT-PCR.
  • the BaEV cell line positive rate detection antibody is a mouse polyclonal antibody of the extracellular region of BaEV, and the fluorescent secondary antibody is rabbit anti-mouse Alexa 647;
  • the lentiviral transfer plasmid containing the coding sequence of the CD19-targeting CAR (PCAR-19B, whose specific information is detailed in Chinese patent: CN107226867A, the full text of which is incorporated herein by reference) and two lentiviral packaging plasmids pMDLg/pRRE , pRSV-Rev (the rightmost column of Table 2), or 293T cells transduced with the three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G respectively (Table 4-5).
  • the virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was detected by flow cytometry. Viruses can be stored at -80°C.
  • the results show that using BaEVRless and VSV-G at the same time can further improve the packaging efficiency compared to using BaEVRless alone (and BaEV-G with the R peptide removed) for lentivirus packaging.
  • Table 1 and Table 4 the results of lentivirus packaging using BaEV-MoRV-tail and BaEV-Rless envelope glycoproteins together with VSV-G show that on the basis of using BaEV-G, VSV- The conclusion that G can improve the packaging efficiency of enveloped viruses is universal.
  • Table 5 it can be seen that the packaging method using BaEVRless and VSV-G is suitable for a variety of promoters, including but not limited to CAG, miniCMV and SV40.
  • the PBNK selected from the above-mentioned lentivirus transduction and activation for 2 days were used to detect the positive rate of CAR-NK, in vitro killing and IFN- ⁇ secretion by flow cytometry 7 days after transduction.
  • the results are shown in Figure 13 and Table 6.
  • the group marked with (2G) represents the group that was simultaneously transfected with pMD2.G plasmid, that is, the group of BaEV::VSV-G chimeric envelope lentivirus, while the group marked with (BaEV) represents the group among which The packaged lentiviral envelope glycoprotein is BaEV and does not contain VSV-G.
  • 293T-BaEVmini indicates that the promoter used for expression of BaEV in the cell line is miniCMV
  • 293T-BaEV indicates that the promoter used for expression of BaEV in the cell line is CAG
  • 293T means that the cell line used is unmodified 293T cells, which are modified to package the lentivirus by introducing the BaEV-G envelope plasmid into the 293T cells.
  • the control group does not package any virus, and only uses unmodified NK cells as a blank control for packaging virus-transduced NK cells in other groups.
  • viruses containing both VSV-G and BaEV-G in the envelope are more effective in immune cells than viruses containing only BaEV-G envelope glycoprotein or variants thereof.
  • Cells that are difficult to transduce have higher infection efficiency (reflected in CAR positivity rate).
  • the CAR-containing immune effector cells constructed by transducing viruses containing VSV-G and BaEV-G all had good killing power (reflected in the killing rate and expression of IFN- ⁇ ), and using BaEV::VSV- Engineered immune effector cells constructed with G chimeric lentivirus have stronger killing power than lentivirus packaged with BaEV-G or its variants.
  • the results of this example also prove that the cell line expressing BaEV-G or its variant protein constructed by lentivirus infection of host cells is similar to the cell line expressing BaEV-G or its variant constructed by transposition. It can be used for efficient lentivirus packaging, and the lentivirus can efficiently transduce immune cells and other cells that are difficult to transduce.
  • the codon-optimized BaEV-Rless coding sequence was ligated into the PB transposon plasmid through double enzyme digestion. After enzyme digestion and sequencing verification, the corresponding plasmid pPBK-CAG-BaEVRless-WPRE was extracted. The concentration of the plasmid should not be lower than 1000ng/ul;
  • the electroporated 293T cells were expanded and cultured, and positive single cells were sorted into a 96-well plate (i.e. 1 cell/well) by flow cytometry.
  • the antibody used for flow cytometry labeling was a mouse polyclonal antibody against the extracellular region of BaEV.
  • the fluorescent secondary antibody is rabbit anti-mouse Alexa 647.
  • the expression abundance of BaEV in different clones detected by flow cytometry after expansion of positive single clones is shown in Figure 14. The abundance is divided into three levels: high, medium and low, as shown in the right panel of Figure 14.
  • the lentiviral plasmid of PCAR-19B (described in detail in patent: CN107226867A) and three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G were transfected into the above-mentioned positive clones by calcium transfer method, and the crude virus extract was used to directly transfect The titer test was performed and the results are shown in Table 7.
  • 293T-BaEV(PB)-1 and 293T-BaEV(PB)-2 are lentiviruses packaged using No. 11 and No. 19 293T-BaEVRless (PB) clones in Table 7, respectively.
  • 293T- BaEV(LV) represents the lentivirus packaged in the 293T cell line expressing BaEVRless constructed using lentiviral transduction of the 293T cell line.
  • 293T represents the lentivirus packaged by direct co-transfection of the BaEVRless packaging plasmid.
  • cell lines expressing BaEV-G and its various variants were constructed by transposition and used to perform slow-motion experiments.
  • Virus packaging can improve packaging efficiency and titer compared to direct transfection of plasmids containing nucleic acids encoding envelope glycoproteins for packaging.
  • Example 7 293T-BaEV-Rless cell line is used to package lentiviruses with different target genes.
  • the genes encoding CD123-CAR, mbIL15, and mbIL12 were ligated into the lentiviral transfer plasmid pBKL2 through double enzyme digestion. After verification by double enzyme digestion and sequencing, pBKL2-CD123-CAR, pBKL2-CD123-CAR, and mbIL12 were obtained respectively. Three plasmids, pBKL2-mbIL15 and pBKL2-mbIL12; perform virus packaging, concentration and titer determination according to the lentivirus packaging method in Example 6. The virus titer is shown in Figure 17. It can be seen that the protocol of this application can be used for lentiviral packaging of various target genes.
  • mbIL12-NK and mbIL15-NK were labeled with IL12-PE (anti-IL12 monoclonal antibody with PE label) and CD215-PE (anti-CD215 monoclonal antibody with PE label) respectively, and CD123-CAR-NK was labeled with rCD123-his+anti His -Detection was performed after APC labeling, and the results are shown in Figure 18. It can be seen that the lentivirus carrying various target genes constructed using this protocol can efficiently construct engineered immune effector cells.
  • ⁇ T cells were activated in vitro for 8 days.
  • the CAR protein was labeled with PE-coupled L protein, and CAR- ⁇ T cells were detected by flow cytometry.
  • Figure 19 (BaEV-Rless:VSV-G chimeric lentivirus and VSV- Comparison of G lentivirus transduction efficiency of ⁇ T cells): Chimeric lentivirus has a higher transduction rate.
  • VSV-G and BaEV-G or variants thereof are used to co-package chimeric envelopes.
  • Viruses can increase the transduction efficiency of the enveloped virus.
  • the 293T-BaEV-Rless cell line was transfected with 10 ⁇ g each of retroviral packaging element-related plasmids pCL-Eco and MSGV1-GFP.
  • the calculation method refers to Example 1.
  • BaEV-Rless and BaEV-Rless::VSV-G chimeric lentivirus packaging, concentration and titer detection were carried out according to the method of Example 6.
  • the titer results are shown in Table 8. The results show that the titer of the chimeric lentivirus The titer is comparable to that of BaEV-Rless.
  • the method of packaging enveloped viruses through cell lines expressing BaEV-G is applicable to various BaEV-G variants (such as BaEV-Rless, BaEV-MoRV-tail, BaEV/TR, etc.), or chimeric enveloped viruses (
  • BaEV-G and its various variants and chimeric enveloped viruses of VSV-G) are examples of various BaEV-G variants (such as BaEV-Rless, BaEV-MoRV-tail, BaEV/TR, etc.), or chimeric enveloped viruses (
  • the packaging of BaEV-G and its various variants and chimeric enveloped viruses of VSV-G) and there is no restriction on the type of target gene being packaged. This method is universal.
  • the detection method please refer to Example 7.
  • the results are shown in Figure 21, as shown below. It can be seen from the figure that in the enveloped virus packaging scheme provided by this application, the use of enveloped viruses packaged with VSV-G and BaEV-G or their variants can further improve the transduction of difficult-to-transduce cells such as immune cells. Guidance efficiency.
  • sequences used in the above examples of the present application are shown in the sequence listing below. It should be understood that the following sequences are only exemplary sequences for the embodiments of the present application, and are not intended to limit any of the embodiments of the present application.
  • the nucleic acid sequence in the following sequence listing may represent a DNA sequence or an RNA sequence. When it represents an RNA sequence, the "T" represents uridine.

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Abstract

L'invention concerne une glycoprotéine membranaire virale ou un polypeptide ou une composition de celle-ci pour l'encapsidation de virus et un virus à membrane encapsidé à l'aide de la glycoprotéine membranaire ou du polypeptide. L'invention concerne également un procédé d'encapsidation d'un virus à membrane à l'aide de la glycoprotéine membranaire virale ou du polypeptide ou de la composition de celui-ci. Le procédé peut améliorer l'efficacité d'encapsidation du virus à membrane et l'efficacité de transduction du virus encapsidé.
PCT/CN2023/107696 2022-07-28 2023-07-17 Glycoprotéine de membrane baev et son utilisation WO2024022147A1 (fr)

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CN202210899789.3A CN117467706A (zh) 2022-07-28 2022-07-28 BaEV囊膜糖蛋白及其应用
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CN202210898270.3A CN117467705A (zh) 2022-07-28 2022-07-28 一种高效的BaEV囊膜病毒包装方法

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