Targeting vectors and their production and use

Abstract

A targeting vector and a method for targeting its host cell are provided. The vector comprises a first molecule that binds to an endocytic receptor of a target cell and a second molecule that promotes the release of a substance carried by the targeting vector into the cytoplasm. When the targeting vector is a viral vector, the first molecule is not part of a viral envelope protein, and the second molecule promotes endosomal or lysosomal escape of the targeting vector. By designing the first molecule based on the endocytic receptor of the target cell, different types of cells can be targeted. After rational mutation of the vector, it is possible to avoid infection of cells that do not require targeting and improve the precision of the vector.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a Chinese patent application filed with the State Intellectual Property Office of the People's Republic of China on September 30, 2022, bearing application number 202211217795.2 and entitled "Targeting vector and its manufacturing method and use," the entire contents of which are incorporated herein by reference in their entirety.

[0002] [Incorporation of sequence listings submitted electronically] This application contains a Sequence Listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The Sequence Listing was created on September 28, 2023, is named "JYSW-PA-PCT-NO-05-1-seql.xml," and has a file size of 1.56 MB. [Technical Field]

[0003] The present invention is in the field of vector delivery and specifically relates to targetable vectors and methods for targeting their host cells. [Background technology]

[0004] Currently, drug delivery mainly employs two methods: (1) viral vectors, such as adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus, and (2) non-viral vectors, such as liposome nanoparticles (LNP), exosomes, virus-like particles (VLP), and antibody-drug conjugates (ADC). Adenovirus, adeno-associated virus, virus-like particles, and antibody-drug conjugates are delivery vectors without a liposome envelope, while retrovirus, lentivirus, nanoliposomes, and exosomes are delivery vectors with a liposome envelope. Enveloped viral vectors target and recognize specific receptor proteins on the cell membrane via the envelope protein. For example, the vesicular stomatitis virus envelope protein (VSVG) can target and recognize the low-density lipoprotein receptor (LDL-R), and the baboon endogenous retrovirus (BaEV) envelope glycoprotein can target and recognize the ASCT1 and ASCT2 receptors, thereby mediating the viral vector's entry into target cells that express the above receptors.

[0005] Currently, vectors based on lentiviral skeletons and VSVG envelope proteins have been widely applied in clinical treatments, such as chimeric antigen receptor T-cell therapy (CAR-T), in which VSVG-carrying lentiviruses package the CAR molecule gene and infect T cells to produce CAR-T.

[0006] Lentiviral vectors are viral vectors modified from human immunodeficiency virus (HIV). They are a type of retrovirus with an RNA genome, in which the toxic gene has been removed and replaced with a foreign gene of interest. They are pseudotyped viruses. They can incorporate foreign genes into the genome using reverse transcriptase for stable expression and are capable of infecting both dividing and non-dividing cells. The original HIV virus carries the gp120 and gp41 complex, which can promote HIV cell infection by recognizing CD4 molecules. Modified lentiviral vectors express the VSVG envelope protein instead of gp120 and gp41. Because VSVG can target and recognize the LDL-R, which is widely expressed, lentiviruses carrying VSVG can widely infect various cell types, including T cells, hepatocytes, cardiomyocytes, neurons, endothelial cells, stem cells, and various tumor cells.

[0007] The advantages of lentiviral vectors are as follows: (1) Long expression time: Lentiviruses can integrate foreign genes into the host cell genome, achieving long-term, stable gene expression that is not lost through cell division and passage. This makes them the first choice for cell experiments and is often used to construct stable cell lines. (2) High safety: Lentiviral vectors are non-pathogenic, and lentiviral vector-modified T cells are used in CAR-T cell therapy. (3) Low immunogenicity: Direct injection into living tissues is unlikely to provoke an immune response, making them suitable for animal experiments. However, their disadvantages include the broad spectrum of LDL-R expression across many types, making them less targetable, and the difficulty of infecting many cells with lentiviruses, such as resting hematopoietic stem cells, resting T cells, NK cells, and B cells.

[0008] The development of targeted lentiviruses has already been reported. For example, (1) CD3 / CD28 antibody-based targeted lentiviruses express CD3 and CD28 antibodies or other antibodies capable of activating T cells with transmembrane sequences on the lentivirus surface, thereby promoting viral particle activation and infection of T cells. This method can effectively promote lentiviral vectors to infect T cells in vivo. While the presence of CD3 antibodies gives this virus a certain targeting ability, the presence of wild-type envelope proteins such as VSVG or Cocal (cocal virus) allows it to still infect other cell types, and its targeting ability still needs to be improved. (2) Fusogen-based targeted viruses. Unlike envelope proteins such as VSVG and BaEV, which simultaneously combine receptor recognition and envelope fusion, the envelope glycoproteins of measles virus (MV) and Nipah virus (NiV) consist of the H / G protein and F protein. The H / G protein is responsible for receptor recognition, and after receptor recognition, the F protein (fusogen) undergoes a conformational change to mediate fusion of the viral envelope with the cell membrane. Linking the H / G protein to antibodies, such as CD4 or CD8 antibodies, can facilitate extracellular delivery of the antibody, allowing the virus to specifically recognize CD4+ or CD8+ T cells. Adding antibodies to the H / G protein increases steric hindrance of the H / G protein, potentially preventing the allosterically activated F protein from reaching the target cell membrane and mediating viral envelope fusion.

[0009] In addition to lentiviruses, AAV vectors are widely used to deliver gene therapy drugs. Adeno-associated viruses are a type of single-stranded DNA virus, and the current scientific consensus is that they do not cause any human diseases. The protein capsid carried by recombinant adeno-associated viruses (rAAV), a gene therapy vector, is almost identical to that of wild-type AAV, but the portion of the genome encoding viral proteins within the capsid has been completely deleted and replaced with a therapeutic gene. The uniquely preserved portion of the AAV genome is the ITRs, which guide genome replication and viral vector assembly. Currently, the majority of AAV clinical trials are focused on four major organs / tissues: the eye, liver, muscle, and central nervous system. Liver-targeted intravenous gene therapy has shown promise for treating metabolic and hematological disorders. Parkinson's disease, hemophilia A, and hemophilia B are the three indications most frequently undergoing clinical trials.

[0010] The advantages of AAV vectors are: (1) they are non-pathogenic, and the vector is derived from a non-pathogenic virus; (2) they have small particles that can penetrate tissues; and (3) they are easy to manufacture and have extremely high packaging titers. Different serotypes of AAV vectors have different infection preferences, but their targeting is still lacking.

[0011] Other vectors, including LNP and ADC drug delivery systems, have already been widely applied in clinical practice.

[0012] Messenger ribonucleic acid (mRNA) encapsulated in LNPs is being used in the production of COVID-19 vaccines, and extensive research is being conducted in the field of tumor vaccines. Because lipid bilayers lack targeting properties, targeting and infecting specific cells is difficult. By linking CD5 antibodies to LNPs, mRNA expressing CAR-T molecules can be targeted and delivered to T cells, thereby producing CAR-T. However, LNPs are naturally easily taken up by dendritic cells (DCs) and macrophages and enriched in the liver. Furthermore, the biomolecular caps formed on LNPs after LNP administration contain hundreds of biomolecules, such as high-density lipoprotein (HDL), which affects their targeting. Therefore, even with antibodies that can be modified for targeting, their targeting remains weak.

[0013] ADC drugs link antibodies to cell-specific endocytic receptors and drugs via a linker. The linker is stable in the blood, preventing drug release during intravenous infusion. However, after the antibody binds to the cell-specific endocytic receptor, it is packaged in lysosomes, where the linker is degraded by lysosomal enzymes, releasing the drug. Because the drug is not degraded by lysosomes, it can exert its effect without relying on lysosomal escape. While ADC drugs have strong targeting properties, it is difficult to deliver drugs other than small molecule toxins, especially biopolymer drugs, to cells using conventional ADC delivery methods because biopolymers such as proteins and nucleic acids are easily degraded by lysosomes.

[0014] In summary, conventional delivery vectors generally have difficulty in achieving targeted delivery to specific cells, and in particular in delivering biological macromolecules to specific cells so that they can exert their functions. Summary of the Invention [Means for solving the problem]

[0015] A first aspect of the present invention provides a targeting vector, which comprises a first molecule that binds to an endocytic receptor of a target cell and a second molecule that promotes release of a substance carried by the targeting vector into the cytoplasm, and when the targeting vector is a viral vector, the first molecule is not part of a viral envelope protein.

[0016] Targeting vectors come in a variety of forms and can be enveloped or non-enveloped vectors, such as retroviral vectors, lentiviral vectors, and lipid nanoparticles, and non-enveloped vectors such as adenoviruses, adeno-associated viruses, and virus-like particles.

[0017] Endocytic receptors are membrane proteins expressed on the cell surface that can induce endocytosis (also known as pinocytosis) after binding to antibodies, ligands, or specific substances. Examples include CD7, CD5, HER2, and mesothelin. Endocytic receptors generally contain domains such as YXXPhi, [D / E]XXXL[L / I], and FXNPXY. However, some membrane proteins, such as CD8 molecules, lack endocytic receptor domains and are generally recognized to have weak endocytic ability. Therefore, screening for endocytic domains often involves screening using CD8 molecules chimerized with random sequences at the intracellular end.

[0018] Different cells express different specific endocytosis receptors, and the first molecule is designed based on the endocytosis receptor of the cell to be targeted. By endocytosing the vector by the target cell via an antibody or ligand (part of the first molecule) that specifically binds to the endocytosis receptor, the vector can be made to have the ability to infect certain cells but not other cells.

[0019] The first molecule binds to an endocytic receptor and then induces receptor-mediated endocytosis. Receptor-mediated endocytosis is a process in which cells specifically take up extracellular proteins or other compounds, depending on cell surface receptors. The cell surface receptors are highly specific and bind to the corresponding ligands to form complexes. The plasma membrane of this area then becomes indented to form coated pits, which then separate from the plasma membrane to form coated vesicles, which take the extracellular material into the cell. After entering the cell, the coated vesicles shed their outer membrane and combine with endosomal vesicles to form large endosomes or endosomes.

[0020] The function of the second molecule is to promote endosomal or lysosomal escape of the targeting vector after endocytosis into the target cell, thereby allowing the targeting vector to enter the cytoplasm. If endosomal or lysosomal escape does not occur after endocytosis, the targeting vector will ultimately be degraded by lysosomes. Therefore, in order to effectively deliver a substance carried by the vector to the cytoplasm, the vector must have the ability to escape from the endosome or the lysosomal membrane.

[0021] Common methods of endosomal / lysosomal escape include: (1) disrupting endosomes / lysosomes, such as using polyethyleneimine to swell and rupture endosomes / lysosomes through the "proton sponge" effect, allowing the loaded material to escape and enter the cytoplasm; (2) reducing the stability of the lysosomal membrane by utilizing the interaction between the positive charges on the vector surface and the negatively charged lysosomal membrane; and (3) achieving escape through membrane fusion between the vector and endosomes / lysosomes, such as modifying the fusion peptide GALA in the vector expression, or incorporating a viral envelope protein with membrane fusion ability, such as VSVG or its mutant, into the vector. In one embodiment, the second molecule is a viral envelope protein and / or a non-viral envelope protein.

[0022] In one specific embodiment, the viral envelope protein is selected from at least one of vesicular stomatitis virus envelope glycoprotein VSVG and its mutants, baboon endogenous retrovirus envelope glycoprotein BaEV and its mutants, feline endogenous retrovirus envelope glycoprotein RD114 and its mutants, and gibbon ape leukemia virus envelope glycoprotein GALV and its mutants, and the non-viral envelope protein is selected from at least one of adeno-associated virus AAV VP1 and its mutants, adeno-associated virus AAV VP2 and its mutants, and polyethyleneimine.

[0023] Viral envelope proteins, such as VSVG, can promote the release of vector-carrying materials by facilitating the fusion of the viral envelope with the endosomal / lysosomal membrane in endosomes / lysosomes. Nonviral envelope proteins, such as AAV VP1 and VP2, undergo conformational changes under acidic conditions, forming perforations in endosomes / lysosomes, thereby releasing vector-carrying materials. Polyethylenimine exerts a proton sponge effect in endosomes / lysosomes; when the pH in the lysosome decreases, PEI captures a large amount of protons, causing an inflow of chloride ions, increasing the osmotic pressure within the endosomes / lysosomes, and ultimately rupturing the endosomes / lysosomes, facilitating the release of vector-carrying materials.

[0024] A variant is a mutant that has at least 75% identity to the non-mutant (wild-type) amino acid sequence, where "at least 75% identity" means 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the non-mutant (wild-type) amino acid sequence.

[0025] Because the VSVG receptor LDL-R is widely expressed in various cells, including activated T cells, hepatocytes, cardiomyocytes, endothelial cells, and stem cells, as well as various tumor cells, pseudotyped lentiviruses carrying VSVG or its variants can infect various cell types. While VSVG-based pseudotyped viruses have broad-spectrum infection capabilities, many cells do not express or express low levels of the VSVG receptor LDL-R, such as NK cells and resting T cells, making them difficult to infect. Furthermore, because LDL-R is widely expressed in various cell types, VSVG-based pseudotyped viruses lack targeting capabilities. For example, various cell types all express LDL-R, making it difficult for lentiviruses to precisely transfect only one type of cell.

[0026] This method utilizes the endosomal / lysosomal escape ability of lentivirus, modifies it, and attaches a first molecule to the lentivirus. The resulting targeting vector can be flexibly applied to different scenarios for different cells. The first molecule is designed based on the cells to be targeted, greatly expanding the scope of application and improving targeting accuracy.

[0027] In one embodiment, the second molecule is selected from VSVG and variants thereof.

[0028] In one embodiment, the first molecule comprises a transmembrane peptide stretch, an antibody or a ligand that binds to an endocytic receptor of a target cell, and in some embodiments, the first molecule further comprises an extracellular hinge region. Membrane-expressed proteins generally require a hinge region to facilitate membrane protein extension, and a commonly used hinge region is the CD8 hinge region.

[0029] The amino acid sequence of the first molecule is not limited as long as it does not affect its function. For example, when the first molecule comprises a CD33 antibody, the first molecule may be a protein that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of the CD33 antibody.

[0030] Endocytic receptors on different target cells vary. Common endocytic receptors include HER2, CD20, CD19, CD79A, CD79B, CD56, CD22, CD138, CD37, CD98, CD309, CD33, CD163, CD163B, CD5, CD7, CD169, CD204, CD205, CD209, CD280, CD302, TROP-2, CD19, NECTIN4, 5T4, CD30, FRα, STEAP1, ENPP3, GCC, SLC44A4, NaPi2b, CA9, SC-16, CD142, P-Cadherin, PSMA, ED-B, endothelin receptors ETB, TN-C, and collagen. IV, Periostin, CEACAM, c-MET, TDGF1, IGF1R, Mesothelin, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, glycosphingolipid, TfR, Gang lioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, ASGPR.

[0031] In one embodiment, the targeting vector is a lentiviral vector, the first molecule expressed by the lentiviral vector is a transmembrane protein, and the second molecule expressed by the lentiviral vector is a viral envelope protein, which has the ability to promote endosomal escape or lysosomal escape. If the target cell does not express a viral envelope protein receptor, the virus can infect the target cell through endocytosis mediated by the first molecule.

[0032] In one specific embodiment, the transmembrane protein is selected from a CD7 antibody, a CD19 antibody, a CD33 antibody, an ASGRP antibody or ligand, a Mesothelin antibody, and a HER2 antibody, and the transmembrane protein has at least about 75%, 80%, 85%, or 90% homology with at least one amino acid sequence selected from the CD8 signal peptide of the CD7 antibody, the TH-69 heavy chain (VH), the GS linker peptide (linker), the TH-69 light chain (VL), the CD8 hinge region, and the CD8 transmembrane region. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to at least one amino acid sequence of the CD8 signal peptide, Gemtuzumab light chain (VL), GS linker peptide, Gemtuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the CD33 antibody. The transmembrane protein may be a protein that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% identical to at least one amino acid sequence of the CD8 signal peptide, FMC-63 heavy chain (VH), GS linker peptide, FMC-63 chain (VL), CD8 hinge region, or CD8 transmembrane region of the CD19 antibody. , 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of at least one of the CD8 signal peptide, ASGPR light chain B11 (VL), GS linker peptide, ASGPR heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the ASGRP antibody, and the transmembrane protein may be at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of at least one of the CD8 signal peptide, ASGPR light chain B11 (VL), GS linker peptide, ASGPR heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the ASGRP antibody.The transmembrane protein may be a protein that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to at least one of the amino acid sequences of the CD8 signal peptide, Pertuzumab light chain (VL), GS linker peptide, Pertuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the HER2 antibody. The transmembrane protein may be a protein that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of at least one of the CD8 signal peptide, PE38 heavy chain (VH), GS linker peptide, PE38 light chain (VL), CD8 hinge region, and CD8 transmembrane region of the MESOTHELIN antibody.

[0033] In one specific example, the viral envelope protein is mutated to weaken its receptor recognition ability, for example, to weaken or eliminate its binding ability to LDL-R, and only retain the ability to escape from the endosome or lysosome, thereby completely relying on the first molecule for targeting. By carrying this mutated envelope protein, the targeting ability of the vector can be further enhanced, allowing it to infect only specific target cells.

[0034] In one specific example, the first molecule comprises an antibody or a ligand, and endocytosis occurs after the antibody binds to the corresponding antigen (endocytosis receptor) on the target cell. The second molecule employs a mutated VSVG, which is mutated by insertion, deletion, or substitution to destroy the binding ability of VSVG to LDL-R but at the same time does not affect the lysosomal escape ability of VSVG. For example, the envelope glycoprotein VSVG of the vesicular stomatitis virus Indiana strain has H8 mutation, N9 mutation, Q10 mutation, K47 mutation, K50 mutation, A51 mutation, etc. The mutations include one or more combinations of mutations at the following positions: S183 mutation, S179 mutation, N180 mutation, I182 mutation, M184 mutation, Y209 mutation, I347 mutation, T350 mutation, T352 mutation, E353 mutation, R354 mutation, deletion of amino acids 1 to 18, deletion of amino acids 19 to 36, deletion of amino acids 37 to 51, deletion of amino acids 314 to 384, deletion of amino acids 321 to 374, deletion of amino acids 331 to 364, deletion of amino acids 344 to 354, and deletion of amino acids 345 to 353.

[0035] Furthermore, VSVG includes one or more combinations of mutations at the following sites: H8 substitution, N9 substitution, Q10 substitution, K47 substitution, K47 deletion, K50 substitution, A51 substitution, S183 substitution, S179 substitution, N180 substitution, I182 substitution, M184 substitution, Y209 substitution, I347 substitution, T350 substitution, T352 substitution, E353 substitution, R354 substitution, deletion of amino acids 1 to 18, deletion of amino acids 19 to 36, deletion of amino acids 37 to 51, deletion of amino acids 314 to 384, deletion of amino acids 321 to 374, deletion of amino acids 331 to 364, deletion of amino acids 344 to 354, and deletion of amino acids 345 to 353.

[0036] Furthermore, K47 and / or R354 of the amino acid sequence of VSVG are mutated, for example, the 47th amino acid of VSVG is substituted from lysine K to glutamine Q, and / or the R354th amino acid is substituted from arginine R to glutamine Q, for example, the 47th amino acid of VSVG is deleted.

[0037] In one specific embodiment, the viral envelope protein is selected from the group consisting of vesicular stomatitis virus Indian strain envelope glycoprotein VSVG, Cocal virus envelope glycoprotein, Maraba virus envelope glycoprotein, Morreton virus envelope glycoprotein, Alagoa virus envelope glycoprotein, New Jersey virus envelope glycoprotein, and Carajas virus envelope glycoprotein.

[0038] In one specific embodiment, when compared across the entire region with the envelope glycoprotein VSVG of the vesicular stomatitis virus Indiana strain, the Cocal virus envelope glycoprotein, Maraba virus envelope glycoprotein, Morreton virus envelope glycoprotein, Alagoa virus envelope glycoprotein, New Jersey virus envelope glycoprotein, and Carajas virus envelope glycoprotein have the following substitutions / deletions: H8 substitution / deletion, N9 substitution / deletion, Q10 substitution / deletion, K47 substitution / deletion, K50 substitution / deletion, A51 substitution / deletion, S183 substitution / deletion, S179 substitution / deletion, N180 substitution / deletion, I182 substitution / deletion, M184 substitution / deletion, Y209 substitution / deletion, I347 substitution / deletion, T35 substitution / deletion, Mutations occur at sites corresponding to the following substitutions / deletions: 0, T352, E353, R354, 1-18 amino acid deletion, 19-36 amino acid deletion, 37-51 amino acid deletion, 314-384 amino acid deletion, 321-374 amino acid deletion, 331-364 amino acid deletion, 344-354 amino acid deletion, and 345-353 amino acid deletion.

[0039] Amino acid mutations include amino acid deletions, substitutions, and insertions. Most research and experiments have focused on amino acid substitutions, while research on amino acid insertions and deletions has remained neglected (Savino S, et al., Insertions and deletions in protein evolution and engineering. Biotechnol Adv. (2022)).

[0040] Substitution or deletion of amino acids at the same site can have unpredictable and quite different effects.

[0041] The 47th amino acid in the extracellular domain of VSV-G is substituted from lysine K to glutamine Q (K47Q), and the 354th amino acid is substituted from arginine R to glutamine Q (R354Q), both of which may weaken or even eliminate the ability of VSV-G to specifically bind to LDL-R. The results that an envelope glycoprotein constructed using VSV-G containing the K47Q or R354Q mutation in the extracellular domain and whose viral envelope contains the first molecule, i.e., a transmembrane protein lentivirus, can still infect target cells specific for the target molecule, do not necessarily predict that an envelope glycoprotein constructed using VSV-G containing a K47 deletion or R354 deletion in the extracellular domain and whose viral envelope contains the first molecule will still have the ability to specifically infect target cells.

[0042] A lentivirus constructed with an envelope glycoprotein containing an R354 deletion in the extracellular domain of VSV-G and containing a membrane-expressing anti-CD7 antibody in the viral envelope was unable to effectively infect CD7+ Jurkat cells, whereas a lentivirus constructed with an envelope glycoprotein containing a K47 deletion in the extracellular domain of VSV-G and containing a membrane-expressing anti-CD7 antibody in the viral envelope was able to effectively infect CD7+ Jurkat cells, further demonstrating that changes in protein function due to amino acid deletions are difficult to predict.

[0043] For example, in lentivirus-infected T cells, the lentivirus envelope expresses mutated VSVG and CD7 antibodies. After the CD7 antibodies bind to the CD7 antigen on the T cell surface, they can mediate endocytosis of the lentivirus. The mutated VSVG mediates lysosomal escape, completing the targeted infection of the lentivirus into T cells. The lentivirus then infects only CD7-expressing T cells and is unable to infect cells that do not express CD7.

[0044] The substance carried by the targeting vector is not limited, and may be a small molecule compound, protein, polypeptide, RNA, or DNA, including, for example, a chimeric antigen receptor (CAR) and a TCR, depending on actual needs. The CAR type is not limited, and the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) containing heavy and light chain variable regions, which specifically bind to a desired antigen. The scFv is selected from monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, human antibodies, nanobodies, and synthetic antibodies. In some embodiments, the CAR further comprises a transmembrane domain (e.g., a CD8 transmembrane domain) and a signaling domain (e.g., CD3ζ) containing one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the CAR comprises one or more costimulatory domains. The type of costimulatory domain is not limited. Indeed, any costimulatory domain known in the art may be used, including but not limited to CD28, 4-1BB, DAP10, DAP12.

[0045] Small molecule compounds are organic compound molecules with a molecular weight of less than 900 daltons, and are particularly small molecules that can be used as drugs.

[0046] In one specific embodiment, the target cells are lymphocytes, bone marrow cells, hematopoietic stem / progenitor cells, or non-blood cells, particularly bone marrow cells, hematopoietic stem / progenitor cells, or non-blood cells, including normal cells and tumor cells.

[0047] In one embodiment, the endocytosis receptor is an endocytosis receptor of bone marrow cells, an endocytosis receptor of hematopoietic stem / progenitor cells, or an endocytosis receptor of non-blood cells.

[0048] In one embodiment, the endocytosis receptor is not CD80, TCR, BCR, CD19, CD20, or IL-13Rα.

[0049] In one embodiment, the endocytosis receptor is not a lymphocyte-specific protein.

[0050] Lymphocyte-specific proteins are proteins specific to B cells, T cells, or NK cells, and T cell-specific proteins include CD3, CD28, CD80, 4-1BB, AhR, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (C XCR4), CD185(CXCR5), CD193(CCR3), CD194(CCR4), CD195(CCR5), CD196(CCR6), CD197(CCR7), CCR10 , PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, IL6ST, gd Contains TCR (TCRγ, TCRδ), Vdeltal, Vdelta2, NKG2D (KLRK1, CD314), TCR (Va24-Jal8), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), and VLA4.

[0051] B cell specific proteins include CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269.

[0052] NK cell-specific proteins include CD56, NKp46, CD16, KIR(s), NKG2 proteins (NKG2D, KLRK1, CD314), KLRB1 (CD161), KLRDl (CD94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R.

[0053] In another aspect of the present invention, a method for producing a targeting vector includes the steps of: designing a first molecule based on an endocytic receptor of a target cell; selecting a second molecule; and b. assembling the first molecule, the second molecule, and the substance carried by the vector to produce a targeting vector.

[0054] In the first case, the first molecule is designed, i.e., modified with an antibody / ligand that can target and recognize a specific receptor on the surface of the virus, and a wild-type viral envelope protein is selected as the second molecule. If the target cell expresses the envelope protein receptor carried by the virus, the presence of the first molecule can promote the infection of the target cell with the targeting vector.

[0055] In the second case, by designing the first molecule, i.e., modifying an antibody / ligand that can target and recognize a specific receptor on the surface of the virus, and selecting a wild-type viral envelope as the second molecule, if the target cell under-expresses or does not express the envelope protein receptor carried by the virus, the first molecule will still allow the virus to complete its infection of the target cell.

[0056] In the third case, the first molecule is designed by modifying an antibody / ligand that can target and recognize a specific receptor on the surface of the virus, and a mutant viral envelope protein that has limited receptor binding but the ability to escape from the endosome / lysosome is selected as the second molecule. This allows the virus to target and infect any target cell, regardless of whether the target cell expresses the envelope protein receptor carried by the virus, and the virus does not infect other cells, greatly improving the targeting accuracy.

[0057] Assembly scheme for enveloped and non-enveloped vectors: To assemble an enveloped vector, for example, using VSVG, a target expression vector can be constructed by mixing a plasmid expressing an antibody / ligand that binds to an endosomal receptor, a lentiviral packaging plasmid, such as psPAX2 and pMD.2G (VSVG or a VSVG mutant), and a lentiviral expression vector.

[0058] For example, the construction of a vector without an envelope can be achieved by linking an antibody / ligand that binds to an endocytic receptor to the AAV envelope protein via a transmembrane peptide stretch, thereby constructing an AAV targeting vector.

[0059] How enveloped and non-enveloped vectors enter cells: Enveloped vectors, such as VSVG-based pseudotyped lentiviral vectors, bind to LDL-R on the cell membrane surface via VSVG and enter cells via clathrin-mediated endocytosis. After endosomes formed by endocytosis are acidified, the VSVG conformation changes, causing the viral envelope to fuse with the endosomal membrane, allowing the virus to escape from the endosome / lysosome and enter the cell nucleus through the nuclear pore.

[0060] For non-enveloped vectors, recombinant AAV viral particles enter cells by binding to glycosylated receptors expressed on the host cell surface and then enter the cells via clathrin-mediated endocytosis. After the endosomes formed by endocytosis are acidified, a conformational change in the VP1 / VP2 portion of the viral capsid causes the virus to detach from the endosome and enter the cell nucleus through the nuclear pore.

[0061] In another embodiment of the present invention, the targeting vector may be used for drug or vaccine delivery, in particular for the delivery of small molecule compounds, proteins, polypeptides, RNA or DNA.

[0062] In another aspect of the invention, there is provided a method of introducing a substance into a cell, said method comprising contacting said cell with a targeting vector.

[0063] In one embodiment, the cells are mammalian cells.

[0064] In one embodiment, the cells are normal cells or cancer cells.

[0065] In one embodiment, the cells are T cells, NK cells, B cells, macrophages, granulocytes, dendritic cells, hematopoietic stem cells, hepatocytes, pancreatic islet cells, nerve cells, and muscle cells.

[0066] In one specific embodiment, the contacting can be performed in vivo or in vitro, for example by intravenous, intraperitoneal, intratumoral, intraosseous or intranodal administration, allowing the targeting vector to enter the target cells in vivo and contact the target cells, or allowing the target virus to directly infect the target cells in vitro.

[0067] The term "mammal" includes any mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, and the like.

[0068] The term "viral envelope protein" refers to naturally occurring viral envelope proteins, such as VSV-G, BaEV, and RD114, which play an important role in viral packaging and infection of host cells.

[0069] Another aspect of the present invention is a composition comprising a targeting vector, which may be used as a medicament, and which may be used in the manufacture of a medicament for gene therapy, immunotherapy, cell therapy, treatment of gene deficiency diseases, treatment of autoimmune diseases, treatment of infectious diseases, and treatment of cancer, including hematological cancers and solid cancers.

[0070] Another aspect of the invention is a method of treating a disease in a subject, said method comprising administering to the subject a therapeutically effective amount of a targeting vector or composition.

[0071] The routes of application / administration of the drug compositions are those common in the art, such as oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebroparenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered via sustained release systems or implanted devices.

[0072] "Treatment" refers to the administration of a therapeutic method described herein to a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a reduction in the rate of cancer cell invasion into surrounding organs, or a reduction in the rate of tumor metastasis or tumor growth). The therapeutic method that effectively treats a patient may vary depending on various factors (e.g., the patient's disease state, age, weight, and the therapeutic method that stimulates the subject's anti-cancer response capabilities).

[0073] As will be appreciated by those skilled in the art, appropriate dosage levels for therapeutic use will vary in part depending on the molecule being delivered, the indication, the route of administration, and the patient's condition (weight, body surface, or organ size) and / or status (age and general health). In some embodiments, the clinician may titrate the dosage or vary the route of administration to obtain the optimal therapeutic effect. [Effects of the Invention]

[0074] This method combines endocytosis with endosomal / lysosomal escape, modifying the vector to have a first molecule capable of binding to the endocytic receptor of the target cell, thereby obtaining a targeting vector, which has the following beneficial effects:

[0075] Based on highly efficient endocytic receptors specific to different cell types, targeting vectors for different cell types can be developed quickly and purposefully without blind screening or trial-and-error validation; By designing the first molecule based on the endocytic receptor of the cell to be targeted, it is possible to target different types of cells, especially cells that do not express or express low levels of the already widely used viral envelope protein receptor (e.g., LDL-R), and the range of applications is wide. After rational mutations have been made to the vector, it is possible to avoid infecting cells that do not require targeting, and the precision can be greatly improved.

[0076] All publications, documents, and patents mentioned herein are specifically incorporated by reference in their entirety, as are each individual publication, document, or patent that is not specifically and individually indicated to be incorporated by reference in its entirety. In the case of conflict, the present application (including any definitions herein) will control. However, any references, articles, publications, patents, patent publications, and patent applications incorporated herein should not be construed as an admission or in any way suggestion that they constitute valid prior art or form part of the common general knowledge of any country in the world.

[0077] The subsection headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [Brief explanation of the drawings]

[0078] [Figure 1] FIG. 1 is a schematic diagram of a targeting vector. [Figure 2] FIG. 1 is a schematic diagram of the pGClenti-GFP lentiviral vector skeleton. [Figure 3] FIG. 1 is a flow diagram of viral infection of peripheral blood NK cells in Example 1. [Figure 4] FIG. 1 is a schematic diagram of the VSVG mutants of Example 2. [Figure 5] FIG. 1 shows the detection of capsid protein P24 in 15 sets of lentiviruses whose envelope glycoproteins are constructed using different VSVG mutants. [Figure 6] FIG. 1 shows titer detection of 15 sets of lentiviruses in which different VSVG mutants are used to construct the envelope glycoprotein. [Figure 7] FIG. 1 is a flow diagram showing how the virus of Example 3 infects CD7+ cells. [Figure 8] FIG. 1 is a flow diagram of infection of Jurkat, Nalm6, and THP1 cells with targeted lentivirus A33-VSV-G-1. [Figure 9]FIG. 1 is a flow diagram of infection of Jurkat and THP1 cells with targeted lentivirus A33-VSV-G-2. [Figure 10] FIG. 1 is a flow diagram of infection of Jurkat and Nalm6 cells with the targeted lentivirus A19-VSV-G-2. [Figure 11] FIG. 1 shows titer detection of targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2. [Figure 12] FIG. 1 is a flow diagram of infection of Jurkat, HuH-7, and Hep-G2 cells with the targeted lentivirus A-ASGPR-VSV-G-2. [Figure 13] FIG. 1 is a flow diagram of infection of Jurkat and MCF7 cells with the targeted lentivirus A-HER-2-VSV-G-2. [Figure 14] FIG. 1 is a flow diagram of the infection of Jurkat and Jurkat-MSN cells with the targeted lentivirus A-Jurkat-MSN-VSV-G-2. [Figure 15] FIG. 1 is a flow diagram of infection of Jurkat cells, Nalm6 cells, and PBMCs with the targeted lentivirus A8-VSV-G-1. [Figure 16] FIG. 1 is a flow diagram of infection of Jurkat cells and PBMCs with targeted lentivirus A8-VSV-G-2. [Figure 17] FIG. 1 is a flow diagram showing the infection of Jurkat cells using lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, respectively. [Figure 18] FIG. 1 is a flow diagram showing the infection of Nalm6 cells with the lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7. DETAILED DESCRIPTION OF THE INVENTION

[0079] Example 1 Construction of NK-targeting lentivirus using wild-type VSVG Because NK cells express the VSVG receptor LDL-R at low levels, conventional VSVG lentiviruses are less likely to infect NK cells. Therefore, the inventors constructed a membrane-expressing CD7 antibody in the lentiviral viral envelope, which binds to CD7 on NK cells to induce lentiviral endocytosis. VSVG then mediates fusion of the viral envelope with the endosomal / lysosomal membrane, resulting in lysosomal escape and further expression of GFP carried by the vector in the cells. The structure of the lentiviral targeting vector is shown in Figure 1. 1 is the first molecule, the CD7 antibody, which can target the endocytic receptor; 2 is the second molecule, VSVG, which mediates endosomal / lysosomal escape.

[0080] 1. Design of membrane-expressed CD7 antibodies The membrane-expressed CD7 antibody sequence includes, from the 5' to the 3' end, a CD8 signal peptide, a TH-69 heavy chain (VH), a GS linker peptide, a TH-69 light chain (VL), a CD8 hinge region, and a CD8 transmembrane region, wherein the CD8 signal peptide amino acid sequence is SEQ ID NO: 1: MALPVTALLLPLALLLHAARP

[0081] The TH-69 heavy chain (VH) amino acid sequence is SEQ ID NO:2: EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS

[0082] The GS linker amino acid sequence is SEQ ID NO:3: GGGGSGGGGSGGGGS

[0083] The TH-69 light chain (VL) amino acid sequence is SEQ ID NO:4: AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR

[0084] The amino acid sequence of the CD8 hinge region is SEQ ID NO:5: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0085] The amino acid sequence of the CD8 transmembrane region is SEQ ID NO:6: IYIWAPLAGTCGVLLLSLVITLYC

[0086] 2. Construction of wild-type VSVG The VSVG wild-type extracellular domain comprises the amino acid sequence shown in SEQ ID NO:7: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0087] 3. Lentivirus encapsulation A lentivirus (skeleton shown in Figure 2) expressing pGClenti-GFP (GFP fluorescent protein) was encapsulated with the psPAX2 plasmid carrying a membrane-tagged CD7 antibody and the pMD2.G (VSVG wild-type) packaging plasmid. Specifically, the four plasmids were mixed and transfected into 293T cells using PEI. The culture supernatant was collected and centrifuged for 48 hours to obtain lentivirus capable of targeting NK cells. When the lentivirus was added to an NK cell culture system, it was able to infect NK cells. The results are shown in Figure 3. The left image shows NK cells infected with VSVG lentivirus expressing GFP, while the right image shows NK cells infected with VSVG lentivirus carrying a membrane-tagged CD7 antibody. While standard VSVG lentivirus has poor NK cell infectivity, the VSVG lentivirus carrying a membrane-tagged CD7 antibody effectively targets and infects NK target cells. This is due to the lack of LDL-R expression and high CD7 expression in NK cells.

[0088] Example 2 Screening of VSVG mutants that lack receptor binding ability but retain endosomal / lysosomal escape ability Based on the binding site of VSVG to LDL-R, the inventors designed a series of VSVG mutants, where Mut represents a mutation and Δ represents a base deletion, as shown in Figure 4, and tested their receptor-binding ability and endosome / lysosome escape ability.

[0089] The 15 sets of VSV-G mutants shown in Figure 4 were used to construct viral envelope proteins, and 15 sets of lentiviruses whose viral envelopes contained membrane-expressing anti-CD7 antibodies were packaged according to the lentivirus packaging method described in Example 1.

[0090] The capsid protein P24 of the 15 sets of lentiviruses obtained by packaging was tested, and the results are shown in Figure 5. Methods for detecting the capsid protein P24 are known to those skilled in the art.

[0091] The 15 sets of lentivirus obtained by packaging were used to infect CD7+ Jurkat cells, respectively, to test the infectivity of each set of lentivirus. The results are shown in FIG.

[0092] As can be seen from Figure 5, the lentiviral capsid protein P24 was detected in all 15 sets of lentivirus obtained by packaging, proving that each set had successfully packaged the lentivirus. However, as can be seen from Figure 6, there were relatively large differences in the biological activity and titer of the 15 sets of lentivirus obtained by packaging.

[0093] As can be seen from Figure 6, the titer of the lentivirus whose viral envelope glycoprotein was constructed using the K47 deletion VSV-G mutant was significantly improved compared to the lentivirus whose viral envelope glycoprotein was constructed using other VSV-G mutants, being approximately twice as high as the titer of the lentivirus whose viral envelope glycoprotein was constructed using the R354Q mutation VSV-G mutant and the K47Q mutation VSV-G mutant. On the other hand, the titer of the lentivirus whose viral envelope glycoprotein was constructed using the R354 deletion VSV-G mutant approached 0, showing almost no infectious ability.

[0094] Example 3 Construction of lentivirus targeting Jurkat cells using mutant VSVG A mutant VSVG was used that retained its ability to escape lysosomes and lost its ability to bind to LDL-R, allowing it to specifically target cells that express CD7; cells that do not express CD7 but express LDL-R were unable to be infected by the lentivirus.

[0095] 1. Design of CD7 antibody This is the same as in the first embodiment.

[0096] 2. Construction of mutated VSVG The VSVG mutant (amino acid 354 mutated to Q) extracellular domain contains the amino acids shown in SEQ ID NO:8: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEQELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0097] 3. Lentivirus encapsulation A targeted lentivirus expressing pGClenti-GFP (GFP fluorescent protein) was packaged with the psPAX2 plasmid carrying a membrane-expressing CD7 antibody and a VSVG mutant packaging plasmid. Specifically, the four plasmids were mixed and transfected into 293T cells using PEI. After 48 hours, the culture supernatant was collected and centrifuged to obtain lentivirus capable of targeting CD7+ cells, including Jurkat cells, CD7+ NK cells, and CD7+ T cells. When the lentivirus was added to the above cell culture system, it was able to infect the corresponding target cells.

[0098] As shown in Figure 7, VSVG lentivirus and targeted lentivirus were used to infect Raji cells, which do not express CD7, and Jurkat cells, which do express CD7. The left column shows the results of infection with the non-targeted lentiviral vector, and the right column shows the results of infection with the targeted lentiviral vector. The control group was Raji cells, which do not express CD7, and the experimental group was CD7 Jurkat cells. The non-targeted lentiviral vector was capable of infecting both Raji and Jurkat cells, while the targeted lentiviral vector was capable of infecting only Jurkat cells, but not Raji cells. The CD7-targeting lentivirus in this example was unable to effectively infect Raji cells and could only effectively infect Jurkat cells because the mutant VSVG used in the targeted lentivirus was incapable of infecting cells, its targeting depended on the CD7 antibody, and it could only target cells that express CD7, whereas the wild-type VSVG carried by the non-targeted lentivirus was able to bind to cells that express LDL-R, regardless of whether these cells expressed CD7.

[0099] Example 4 Construction of lentivirus targeting CD33+ cells 1. Design of membrane-expressed CD33 antibodies The membrane-expressed CD33 antibody sequence contains, from the 5' to 3' end, a CD8 signal peptide, a Gemtuzumab light chain (VL), a GS linker peptide, a Gemtuzumab heavy chain (VH), a CD8 hinge region, and a CD8 transmembrane region.

[0100] The Gemtuzumab light chain (VL) amino acid sequence is SEQ ID NO:9: DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVE VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0101] The Gemtuzumab heavy chain (VH) amino acid sequence is SEQ ID NO:10: EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0102] 2. Construction of mutant VSV-G with reference to Example 3 Referring to Example 3, a mutant VSV-G was constructed, and the VSV-G mutant extracellular domain contained the amino acid sequence shown in SEQ ID NO:8 (the 354th amino acid, arginine, was mutated to glutamine Q, forming mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO:21 (the 47th amino acid, lysine K, was deleted, forming mutant VSV-G-2).

[0103] 3. Lentivirus encapsulation Referring to Example 3, targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 were packaged and used to infect CD33+ and CD33- cells, respectively.

[0104] 0.5 x 10 each 6 Jurkat cells, Nalm6 cells, and THP1 cells were resuspended in 200 μL of medium containing 1640 (Brand: Elgbio, Product Number: EH80809) medium and 10% FBS (Brand: Excell, Product Number: FSP500). The lentivirus A33-VSV-G-1 targeting CD33+ cells was added to each cell culture system at an MOI of 1. The cells were mixed thoroughly and cultured in an incubator at 37°C. GFP expression in Jurkat cells, Nalm6 cells, and THP1 cells was detected on Day 2. The results are shown in Figure 8.

[0105] As can be seen in Figure 8, the targeted lentivirus A33-VSV-G-1 was unable to effectively infect Jurkat and Nalm6 cells, which express LDL-R but not CD33, but was able to effectively infect THP1 cells, which express CD33.

[0106] Refer to the method described above for infecting Jurkat cells and THP1 cells with the targeted lentivirus A33-VSV-G-1, and infect 1 x 10 cells, respectively. 5 Jurkat cells and THP1 cells were taken, and the targeted lentivirus A33-VSV-G-2 was added to the Jurkat cell culture system and THP1 cell culture system at an MOI of 1, respectively. The GFP expression status in each set of culture system was detected on day 2, and the results are shown in Figure 9.

[0107] As can be seen in Figure 9, the targeted lentivirus A33-VSV-G-2 was unable to effectively infect Jurkat cells that express LDL-R but not CD33, but was able to effectively infect THP1 cells that express CD33.

[0108] In this example, the viral envelope of the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 constructed using the mutant VSV-G-1 or VSV-G-2 contains a membrane-expressing anti-CD33 antibody, which can specifically bind to the endocytic receptor CD33 on the surface of CD33+ cells, and enter and infect CD33+ cells through endocytosis. Since the viral envelope glycoproteins are constructed using mutant VSV-G-1 or VSV-G-2, the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 have a weakened or lost ability to specifically bind to LDL-R, and are unable to effectively infect cells expressing LDL-R. This effectively improves the targeting ability of the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 to infect CD33+ cells.

[0109] Flow cytometry antibody used for CD33 detection by flow cytometry: Product name: APC-CD33, Brand: Biolegend, Product number: #366606.

[0110] Example 5 Construction of lentivirus targeting CD19+ cells 1. Design of membrane-expressed CD19 antibody The membrane-expressed CD19 antibody sequence contains, from the 5' to 3' end, a CD8 signal peptide, an FMC-63 heavy chain (VH), a GS linker peptide, an FMC-63 light chain (VL), a CD8 hinge region, and a CD8 transmembrane region.

[0111] The FMC-63 heavy chain (VH) amino acid sequence is as shown in SEQ ID NO:11.

[0112] The FMC-63 light chain (VL) amino acid sequence is as shown in SEQ ID NO:12.

[0113] 2. Construction of mutant VSV-G with reference to Example 3 Referring to Example 3, a mutant VSV-G was constructed, and the VSV-G mutant extracellular domain contained the amino acid sequence shown in SEQ ID NO:8 (the 354th amino acid, arginine, was mutated to glutamine Q, forming mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO:21 (the 47th amino acid, lysine K, was deleted, forming mutant VSV-G-2).

[0114] 3. Lentivirus encapsulation Referring to Example 3, the targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 were constructed using the mutant VSV-G-1 and mutant VSV-G-2.

[0115] 1 x 10 each 5 100 CD19- Jurkat cells and 100 CD19+ Nalm6 cells were resuspended in 200 μL of medium containing 1640 (Brand: Elgbio, Product No.: EH80809) medium and 10% FBS (Brand: Excell, Product No.: FSP500). The lentiviruses A19-VSV-G-1 and A19-VSV-G-2 targeted to CD19+ cells were added to each set of cell culture system at an MOI of 1, mixed evenly, and cultured in a 37°C incubator. Day 1 2. The expression status of GFP in Jurkat cells and Nalm6 cells was detected. The results of infection of Jurkat cells and Nalm6 cells with the targeted lentivirus A19-VSV-G-2 are shown in Figure 10, and the titer detection results of the targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 are shown in Figure 11.

[0116] As can be seen from Figure 10, the targeted lentivirus A19-VSV-G-2 was unable to effectively infect Jurkat cells that do not express CD19, but was able to effectively infect Nalm6 cells that express CD19.

[0117] The targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 specifically bound to the endocytic receptor CD19 on the surface of Nalm6 cells via the anti-CD19 antibody contained in their viral envelope, and then entered and infected CD19+ Nalm6 cells through endocytosis.

[0118] As can be seen from Figure 11, the titer of the targeted lentivirus A19-VSV-G-2, whose envelope glycoprotein is constructed using the mutant VSV-G-2 (K47 deletion), was significantly improved compared to the targeted lentivirus A19-VSV-G-1, whose envelope glycoprotein is constructed using the mutant VSV-G-1 (R354Q mutation).

[0119] Flow cytometry antibody used for CD19 detection by flow cytometry: Product name: APC-CD19, Brand: Si'anchi, Product number: #S0098.

[0120] Example 6 Construction of lentivirus targeting asialoglycoprotein receptor-positive (ASGPR+) hepatocytes

[0121] The asialoglycoprotein receptor (ASGPR) to be targeted may be N-acetylgalactosamine (GalNAc) or an ASGPR antibody. GalNAc can be added to the surface of the lentivirus envelope by modification, or an ASGPR antibody can be expressed on the membrane to produce a lentivirus that targets liver cells.

[0122] 1. Design of membrane-expressed ASGPR antibodies The membrane-expressed CD33 antibody sequence contains, from the 5' to 3' end, a CD8 signal peptide, ASGPR light chain B11 (VL), a GS linker peptide, ASGPR heavy chain (VH), a CD8 hinge region, and a CD8 transmembrane region.

[0123] The ASGPR antibody light chain (VL) amino acid sequence is SEQ ID NO:13: DIVLTQPPSASGTPGQRVTISCTGSSSGIGNAYVSWYQQLPGKAPKLLIYKNGQRPSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKVTVL The ASGPR antibody heavy chain (VH) amino acid sequence is SEQ ID NO:14: MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSAITTGGGSPNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRTAGYFDYWGQGALVTVSSGSA 2. Construction of mutant VSV-G with reference to Example 3 Referring to Example 3, a mutant VSV-G-2 was constructed, and the extracellular domain of the mutant VSV-G-2 comprises the amino acid sequence shown in SEQ ID NO:21.

[0124] 3. Lentivirus encapsulation Referring to Example 3, a lentivirus A-ASGPR-VSV-G-2 targeted with the mutant VSV-G-2 was constructed.

[0125] 1 x 10 each 5ASGPR- Jurkat cells, ASGPR+ HuH-7 cells, and ASGPR+ Hep-G2 cells were resuspended in 200 μL of culture medium containing DMEM medium and 10% FBS (DMEM, brand: Gibco, product number: C12430500BT, FBS, brand: Gibco, product number: FSP500). The lentivirus A-ASGPR-VSV-G-2 targeting ASGPR+ cells was added to each cell culture system at an MOI of 1, mixed thoroughly, and cultured in a 37°C incubator. On day 2, GFP expression in Jurkat cells, HuH-7 cells, and Hep-G2 cells was detected. The results are shown in Figure 12.

[0126] As can be seen from Figure 12, the targeted lentivirus A-ASGPR-VSV-G-2 was unable to effectively infect Jurkat cells that do not express ASGPR, but was able to effectively infect HuH-7 and Hep-G2 cells that express ASGPR.

[0127] The targeted lentivirus A-ASGPR-VSV-G-2 specifically bound to the endocytic receptor ASGPR on the surface of ASGPR+ cells via the anti-ASGPR antibody contained in its viral envelope, and then entered and infected ASGPR+ HuH-7 and Hep-G2 cells through endocytosis.

[0128] Flow cytometry antibody used for ASGPR detection by flow cytometry: Product name: PE-ASGPR1, Brand: BD, Product number: #563655.

[0129] Example 7 Construction of lentivirus targeting HER2+ cells 1. Design of membrane-expressed HER2 antibody The membrane-expressed HER2+ antibody sequence contains, from the 5' to 3' end, a CD8 signal peptide, Pertuzumab light chain (VL), a GS linker peptide, Pertuzumab heavy chain (VH), a CD8 hinge region, and a CD8 transmembrane region.

[0130] The Pertuzumab light chain (VL) amino acid sequence is SEQ ID NO:15: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0131] The Pertuzumab heavy chain (VH) amino acid sequence is SEQ ID NO:16: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0132] 2. Construction of mutant VSV-G with reference to Example 3 Referring to Example 3, a mutant VSV-G-2 was constructed, and the extracellular domain of the mutant VSV-G-2 comprises the amino acid sequence shown in SEQ ID NO:21.

[0133] 3. Lentivirus encapsulation Referring to Example 3, a lentivirus A-HER-2-VSV-G-2 targeted with the mutant VSV-G-2 was constructed.

[0134] 1 x 10 each 5 100 HER-2- Jurkat cells and HER-2+ MCF7 cells were resuspended in 200 μL of culture medium containing DMEM medium and 10% FBS (DMEM, brand: Gibco, product number: C12430500BT, FBS, brand: Gibco, product number: FSP500). The lentivirus A-HER-2-VSV-G-2 targeted to HER-2+ cells was added to each set of cell culture system at an MOI of 1, mixed evenly, and cultured in an incubator at 37°C. On day 2, the expression of GFP in Jurkat cells and MCF7 cells was detected. The results are shown in Figure 13.

[0135] As can be seen in Figure 13, the targeted lentivirus A-HER-2-VSV-G-2 was unable to effectively infect Jurkat cells that do not express HER-2, but was able to effectively infect MCF7 cells that express HER-2.

[0136] The targeted lentivirus A-HER-2-VSV-G-2 specifically bound to the endocytic receptor HER-2 on the surface of MCF7 cells via the anti-HER-2 antibody contained in its viral envelope, and then entered and infected HER-2+ MCF7 cells through endocytosis.

[0137] Flow cytometry antibody used for HER-2 detection by flow cytometry: Product name: APC-CD340(HER-2), Brand: Biolegend, Product number: #324407.

[0138] Example 8 Construction of lentivirus targeting MESOTHELIN+ cells 1. Design of membrane-expressed mesothelin antibodies The membrane-expressed MESOTHELIN+ (MSN) antibody sequence contains, from the 5' to 3' end, a CD8 signal peptide, a PE38 heavy chain (VH), a GS linker peptide, a PE38 light chain (VL), a CD8 hinge region, and a CD8 transmembrane region.

[0139] The PE38 heavy chain (VH) amino acid sequence is SEQ ID NO:17: MQVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGV

[0140] The PE38 light chain (VL) amino acid sequence is SEQ ID NO:18: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIK

[0141] 2. Construction of mutant VSV-G with reference to Example 3 Referring to Example 3, a mutant VSV-G-2 was constructed, and the extracellular domain of the mutant VSV-G-2 comprises the amino acid sequence shown in SEQ ID NO:21.

[0142] 3. Lentivirus encapsulation Referring to Example 3, a lentivirus A-MSN-VSV-G-2 targeted with the mutant VSV-G-2 was constructed.

[0143] 4. Construction of Jurkat-MSN overexpressing cell line A conventional lentivirus whose envelope glycoprotein is wild-type VSV-G is used to carry nucleic acid encoding MSN, which is then used to infect a Jurkat cell line to construct a Jurkat-MSN cell line that overexpresses MSN. Methods for constructing overexpressing cell lines are known to those skilled in the art.

[0144] 1 x 10 each 5 200 μL of MSN- Jurkat cells and MSN+ Jurkat-MSN cells were resuspended in 200 μL of culture medium containing 1640 + 10% FBS (1640, brand: Elgbio, product number: #EH80809, FBS, brand: Yikes, product number: #FSP500). The lentivirus A-MSN-VSV-G-2 targeted to MSN+ cells was added to each set of cell culture system at an MOI of 1, mixed evenly, and cultured in an incubator at 37°C. On day 2, the expression of GFP in Jurkat cells and Jurkat-MSN cells was detected. The results are shown in Figure 14.

[0145] As can be seen from Figure 14, the targeted lentivirus A-Jurkat-MSN-VSV-G-2 was unable to effectively infect Jurkat cells that did not express MSN, but was able to effectively infect Jurkat-MSN cells that expressed MSN.

[0146] The targeted lentivirus A-MSN-VSV-G-2 specifically bound to the endocytic receptor MSN on the surface of Jurkat-MSN cells via the anti-MSN antibody contained in its viral envelope, and then entered and infected MSN+ Jurkat-MSN cells through endocytosis.

[0147] Flow cytometry antibody used for detecting MSNs by flow cytometry: Product name: APC-Mesothelin, Brand: R&D, Product number: #FAB32652A.

[0148] Example 9 Construction of a non-endocytic receptor CD8 antibody-based lentivirus targeting CD8+ cells 1. Design of membrane-expressed CD8 antibodies The membrane-expressed CD8+ antibody sequence contains, from the 5' to 3' end, a CD8 signal peptide, a CD8 antibody heavy chain (VH), a GS linker peptide, a CD8 antibody light chain (VL), a CD8 hinge region, and a CD8 transmembrane region.

[0149] The CD8 antibody heavy chain (VH) amino acid sequence is SEQ ID NO:19: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS

[0150] The CD8 antibody light chain (VL) amino acid sequence is SEQ ID NO:20: DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIK

[0151] 2. Construction of mutant VSV-G with reference to Example 3 Referring to Example 3, a mutant VSV-G was constructed, and the extracellular domain of the VSV-G mutant contained the amino acid sequence shown in SEQ ID NO:8 (the 354th amino acid was mutated to Q, forming mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO:21 (the 47th amino acid, lysine K, was deleted, forming mutant VSV-G-2).

[0152] 3. Lentivirus encapsulation Referring to Example 3, the mutant VSV-G-1 or mutant VSV-G-2 was used to package the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2, whose viral envelope contains a membrane-expressing anti-CD8 antibody.

[0153] 1 x 10 each 5 Jurkat cells, Nalm6 cells, and PBMCs were each resuspended in 200 μL of medium. The medium for resuspending the Jurkat cells and Nalm6 cells contained 1640 medium (brand: Elgbio, product number: EH80809) and 10% FBS (brand: EXCELL, product number: FSP500), and the medium for resuspending the PBMCs contained XVT medium (product name: PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), product number: 91154), IL-7 (product name: IL-7 Protein, Human, Recombinant, brand: Yiqiao Shenzhou, product number: 11821-HNAE) at a final concentration of 20 ng / mL, and IL-15 (product name: IL-15 Protein, Human, Recombinant (His Tag), brand: Yiqiao Shenzhou, product number: 10360-H07E) at a final concentration of 20 ng / mL.

[0154] The targeted lentivirus A8-VSV-G-1 was added to the Jurkat cells, Nalm6 cells, and PBMCs cell culture systems at an MOI of 1, mixed evenly, and cultured in an incubator at 37°C. On day 2, the GFP expression status in the Jurkat cells, Nalm6 cells, and PBMCs was detected. The results are shown in Figure 15.

[0155] As can be seen from Figure 15, the targeted lentivirus A8-VSV-G-1 constructed using mutant VSV-G-1 was unable to effectively infect Jurkat and Nalm6 cells, which express LDL-R but do not express CD8, nor was it able to effectively infect PBMCs, which express CD8.

[0156] 1 x 10 each 5 Jurkat cells and PBMCs were taken, and the targeted lentivirus A8-VSV-G-2 was added to the Jurkat cell culture system and the PBMC culture system at an MOI of 1, according to the method described above, by uniformly mixing and culturing each set of cells with the targeted lentivirus A8-VSV-G-1. On day 2, the GFP expression status in the Jurkat cells and PBMCs was detected. The results are shown in Figure 16.

[0157] As can be seen from Figure 16, the targeted lentivirus A8-VSV-G-2 was unable to effectively infect Jurakt cells, which express LDL-R but do not express CD8, nor was it able to effectively infect PBMCs, which express CD8.

[0158] After CD8+ cells were exposed to the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2, the membrane-expressed anti-CD8 antibody contained in the viral envelope of the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2 specifically bound to the receptor CD8 on the surface of CD8+ cells in PBMCs. However, because CD8 is a non-endocytic receptor and cannot efficiently mediate endocytosis, the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2 could not effectively enter and infect CD8+ cells.

[0159] The results showed that after contact between CD8+ cells and lentivirus, the CD8 receptor could bind to the antibody but could not be effectively endocytosed, and the membrane-expressed CD8 antibody-based lentiviral vector could not effectively mediate infection of CD8+ cells. Therefore, receptors without efficient endocytosis were not suitable as the first molecule for constructing the targeting vector of the present invention.

[0160] Flow cytometry antibody used for CD8 detection by flow cytometry: APC-CD8, brand: BD, product number: 566852.

[0161] Example 10 Comparison of infection efficiency of lentiviruses containing K47 deletion or R354 deletion in the VSV-G extracellular domain

[0162] An envelope plasmid carrying a nucleic acid encoding the membrane-expressed anti-CD7 antibody described in Example 1 and a nucleic acid encoding VSV-G containing a K47 deletion in the extracellular domain (K47 deletion-A7 envelope plasmid), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a lentiviral GFP plasmid were prepared. The K47 deletion-A7 envelope plasmid was synthesized by conventional molecular cloning methods. The lentivirus dK47-VSV-G-A7 is packaged and produced according to the packaging method for packaging and producing the lentivirus described in Example 3. Of the four plasmids mentioned above, the envelope plasmid containing a K47 deletion in the VSV-G extracellular domain is replaced with an envelope plasmid containing an R354 deletion, K47Q, or R354Q mutation in the VSV-G extracellular domain, and the lentivirus dR354-VSV-G-A7, sK47Q-VSV-G-A7, or sR354Q-VSV-G-A7 containing an R354 deletion, K47Q, or R354Q mutation in the VSV-G extracellular domain is packaged and produced according to the packaging method for the lentiviral vector dK47-VSV-G-A7 mentioned above. The VSV-G extracellular domain containing the K47 deletion in the extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 21, The VSV-G extracellular domain containing the R354 deletion in the extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 22, The VSV-G extracellular domain containing the K47Q mutation in the extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 23, The VSV-G extracellular domain containing the R354Q mutation comprises the amino acid sequence shown in SEQ ID NO:8.

[0163] Each of the four lentiviruses was added to four sets of CD7+ Jurkat cells at an MOI of 1, mixed thoroughly, and allowed to infect at room temperature for 10 minutes. After that, 10 mL of DPBS buffer was added, mixed thoroughly, and centrifuged at 500 g for 3 minutes. The supernatant was then aspirated and discarded. Each of the four sets of CD7+ Jurkat cells was resuspended in 1 mL of 1640 medium (Brand: ELGBIO, Product Number: #EH80809; FBS serum: Brand: EXCELL, Product Number: #FSP500) containing 10% FBS. The four sets of CD7+ Jurkat cells were then cultured in an incubator at 37°C with 5% CO2. GFP expression was detected on Day 2. The results are shown in Figure 17.

[0164] As can be seen from Figure 17, the lentiviruses dK47-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, which contain K47 deletion, R354Q, or K47Q mutation in the VSV-G extracellular domain, can all specifically bind to the endocytic receptor CD7 expressed by Jurkat cells and can effectively infect CD7+ Jurkat cells through endocytosis, while the lentivirus dR354-VSV-G-A7, which contains R354 deletion in the VSV-G extracellular domain, is less able to infect CD7+ Jurkat cells through endocytosis. This indicates that after the R354 deletion in the extracellular domain of VSV-G, an unpredictable and unknown change occurred in the function of VSV-G, unlike the K47 deletion. Even though the viral envelope contained membrane-expressed anti-CD7 antibodies, the lentivirus dR354-VSV-G-A7 still lost the ability to infect CD7+ cells.

[0165] Referring to the above virus infection and in vitro culture methods, the above four lentiviruses, dK47-VSV-G-A7, sK47Q-VSV-G-A7, sR354Q-VSV-G-A7, and dR354-VSV-G-A7, were added to four sets of CD7-Nalm6 cells at an MOI of 1, respectively, and the cells were cultured in an incubator at 37°C with 5% CO2. GFP expression was detected on day 2, and the results are shown in Figure 18.

[0166] As can be seen from Figure 18, none of the lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7 were able to infect CD7- Nalm6 cells, indicating that the K47 deletion in the VSV-G extracellular domain does not affect the targeting ability of the lentivirus dK47-VSV-G-A7 to infect CD7+ cells.

[0167] As described above, the present invention is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, and any modifications or replacements that can be easily conceived by any person skilled in the art within the technical scope revealed by the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined based on the scope of the claims.

Claims

1. A targeting vector comprising a first molecule that binds to an endocytic receptor of a target cell and a second molecule that promotes the release of a substance carried by the targeting vector into the cytoplasm, wherein when the targeting vector is a viral vector, the first molecule is not part of a viral envelope protein.

2. The targeting vector of claim 1, wherein the second molecule promotes endosomal or lysosomal escape in the targeting vector.

3. The targeting vector according to claim 1 , wherein the targeting vector is an enveloped or non-enveloped vector.

4. The targeting vector of claim 3, wherein the enveloped vector is selected from a retroviral vector, a lentiviral vector, and a lipid nanoparticle, and the non-enveloped vector is selected from an adenovirus, an adeno-associated virus, and a virus-like particle.

5. The targeting vector of claim 1, wherein the second molecule is a viral envelope protein and / or a non-viral envelope protein.

6. The viral envelope proteins include vesicular stomatitis virus envelope glycoprotein VSVG and its mutants, Cocal virus envelope glycoprotein and its mutants, Maraba virus envelope glycoprotein and its mutants, Morreton virus envelope glycoprotein and its mutants, Alagoa virus envelope glycoprotein and its mutants, New The targeting vector of claim 5, wherein the non-viral envelope protein is selected from at least one of Jersey virus envelope glycoprotein and variants thereof, Carajas virus envelope glycoprotein and variants thereof, baboon endogenous retrovirus envelope glycoprotein BaEV and variants thereof, feline endogenous retrovirus envelope glycoprotein RD114 and variants thereof, and gibbon ape leukemia virus envelope glycoprotein GALV and variants thereof, and the non-viral envelope protein is selected from at least one of adeno-associated virus AAV VP1 and variants thereof, adeno-associated virus AAV VP2 and variants thereof, and polyethyleneimine.

7. The targeting vector of claim 5, wherein the second molecule is selected from VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof.

8. 2. The targeting vector of claim 1, wherein the first molecule comprises a transmembrane peptide stretch, an antibody or a ligand that binds to an endocytic receptor of the target cell.

9. The targeting vector of claim 8, wherein the first molecule further comprises an extracellular hinge region.

10. The endocytic receptors include HER2, CD20, CD19, CD79A, CD79B, CD56, CD22, CD138, CD37, CD98, CD309, CD33, CD163, CD163B, CD5, CD7, CD169, CD204, CD205, CD209, CD280, CD302, TROP-2, CD19, NECTIN4, 5T4, CD30 TROP2, FRα, STEAP1, ENPP3, GCC, SLC44A4, NaPi2b, CA9, SC-16, CD142, P-Cadherin, PSMA, ED-B, endothelin receptors ETB, TN-C, and collagen. IV, Periostin, CEACAM, c-MET, TDGF1, IGF1R, Mesothelin, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, glycosphingolipid, TfR, Ganglioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, and ASGPR.

11. The targeting vector of claim 1, wherein the targeting vector is a lentiviral vector, a first molecule expressed by the lentiviral vector is a transmembrane protein, and a second molecule expressed by the lentiviral vector is a viral envelope protein.

12. The targeting vector of claim 11, wherein the viral envelope protein is mutated to weaken its receptor recognition ability.

13. The targeting vector of claim 12, wherein the mutation is a deletion or substitution.

14. The targeting vector of claim 12 or 13, characterized in that the viral envelope protein is selected from vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG, Cocal virus envelope glycoprotein, Maraba virus envelope glycoprotein, Morreton virus envelope glycoprotein, Alagoa virus envelope glycoprotein, New Jersey virus envelope glycoprotein, and Carajas virus envelope glycoprotein.

15. The vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG contains an H8 substitution / deletion, an N9 substitution / deletion, a Q10 substitution / deletion, a K47 substitution / deletion, a K50 substitution / deletion, an A51 substitution / deletion, an S183 substitution / deletion, an S179 substitution / deletion, an N180 substitution / deletion, an I182 substitution / deletion, an M184 substitution / deletion, a Y209 substitution / deletion, an I347 substitution / deletion, a T350 substitution / deletion, a T352 substitution / deletion, an E35 The targeting vector of claim 14, comprising one or more combinations of mutations at the following sites: substitution / deletion of R3, substitution of R354, deletion of amino acids at positions 1 to 18, deletion of amino acids at positions 19 to 36, deletion of amino acids at positions 37 to 51, deletion of amino acids at positions 314 to 384, deletion of amino acids at positions 321 to 374, deletion of amino acids at positions 331 to 364, deletion of amino acids at positions 344 to 354, and deletion of amino acids at positions 345 to 353.

16. When compared across the entire region with the vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG, the Cocal virus envelope glycoprotein, Maraba virus envelope glycoprotein, Morreton virus envelope glycoprotein, Alagoa virus envelope glycoprotein, New Jersey virus envelope glycoprotein, and Carajas virus envelope glycoprotein have the following mutations: H8 substitution / deletion, N9 substitution / deletion, Q10 substitution / deletion, K47 substitution / deletion, K50 substitution / deletion, A51 substitution / deletion, S183 substitution / deletion, S179 substitution / deletion, N180 substitution / deletion, I182 substitution / deletion, M184 substitution / deletion, Y209 substitution / deletion, I347 substitution / deletion, T350 substitution / deletion, and T352 substitution. The targeting vector of claim 14, wherein mutations occur at sites corresponding to the amino acid deletion / substitution of E353, the substitution of R354, the deletion of amino acids 1 to 18, the deletion of amino acids 19 to 36, the deletion of amino acids 37 to 51, the deletion of amino acids 314 to 384, the deletion of amino acids 321 to 374, the deletion of amino acids 331 to 364, the deletion of amino acids 344 to 354, and the deletion of amino acids 345 to 353.

17. The targeting vector of claim 11, wherein the transmembrane protein is a CD7 antibody, a CD19 antibody, a CD33 antibody, an ASGRP antibody or ligand, a mesothelin antibody, or a HER2 antibody.

18. The targeting vector according to claim 1, wherein the substance is at least one of a small molecule compound, a protein, a polypeptide, RNA, and DNA.

19. The targeting vector of claim 1, wherein the target cells are lymphocytes, bone marrow cells, hematopoietic stem / progenitor cells, or non-blood cells.

20. The targeting vector of claim 1, wherein the endocytic receptor is not a lymphocyte-specific protein.

21. A method for producing the targeting vector according to any one of claims 1 to 20, comprising: designing a first molecule based on an endocytic receptor of a target cell; selecting a second molecule; and assembling the first molecule, the second molecule, and a substance carried by the vector to produce a targeting vector.

22. Use of the targeting vector according to any one of claims 1 to 20 in drug or vaccine delivery.

23. The targeting vector according to any one of claims 1 to 20, which is used for delivery of a small molecule compound, a protein, a polypeptide, RNA or DNA.

24. A method for introducing a substance into a cell, the method comprising contacting the cell with the targeting vector of any one of claims 1 to 20.

25. 25. The method of claim 24, wherein the cell is a mammalian cell.

26. 25. The method of claim 24, wherein the cell is a normal cell or a cancer cell.

27. 25. The method of claim 24, wherein the cell is a T cell, NK cell, B cell, macrophage, granulocyte, dendritic cell, hematopoietic stem cell, hepatocyte, pancreatic islet cell, neuron, or muscle cell.

28. 25. The method of claim 24, wherein the contacting is performed in vivo or in vitro.

29. A composition comprising the targeting vector of any one of claims 1 to 20.

30. 30. The composition of claim 29 for use as a medicament.

31. 31. The composition of claim 30, for use in gene therapy, immunotherapy, cell therapy, treatment of gene deficiency diseases, treatment of autoimmune diseases, treatment of infectious diseases, and treatment of cancer, including hematological cancers and solid cancers.

32. A method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the targeting vector according to any one of claims 1 to 20 or the composition according to any one of claims 29 to 31.

Images