Targeting carrier and preparation method and application thereof

Abstract

The invention provides a targeting vector and a method for targeting a host cell, the vector comprises a first molecule combined with an endocytosis receptor of a target cell and a second molecule for promoting release of a substance carried by the targeting vector into cytoplasm, when the targeting vector is a virus vector, the first molecule is not a part of a virus envelope protein, and the second molecule is not a part of a virus envelope protein. The second molecules promote endosome escape or lysosome escape of the targeting carrier. According to the present invention, the first molecule is designed according to the endocytosis receptor of the to-be-targeted cell so as to target different types of cells, and after the vector is subjected to reasonable mutation, the infection of the cells not requiring the targeting can be avoided, and the accuracy of the vector can be improved.

Description

Targeting vector and its preparation method and use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on September 30, 2022, with application number 202211217795.2 and invention name “Targeting vector and its preparation method and use”, the entire contents of which are incorporated herein by reference in their entirety.

[0003] References to sequence listings submitted electronically

[0004] This application includes a sequence listing in XML format submitted electronically, 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-seq1.xml", and has a file size of 1.56 MB. Technical Field

[0005] The present invention belongs to the field of vector delivery, and in particular relates to a targeted vector and a method for targeting host cells thereof. Background Art

[0006] Currently, drug delivery is mainly achieved through two methods: (1) viral vectors, such as adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus, and (2) non-viral vectors, such as liposome nanoparticles (LNPs), exosomes, virus-like particles (VLPs), and antibody-drug conjugates (ADCs). Among them, adenovirus, adeno-associated virus, virus-like particles, and antibody-drug conjugates are delivery vectors without liposome envelopes, while retrovirus, lentivirus, nanoliposomes, and exosomes are delivery vectors with liposome envelopes. Enveloped viral vectors target specific receptor proteins on the cell membrane through their envelope proteins, such as the vesicular stomatitis virus envelope protein (VSVG) that can target the low-density lipoprotein receptor (LDL-R), and the baboon endogenous retrovirus (BaEV) envelope glycoprotein that can target the ASCT1 and ASCT2 receptors, thereby mediating the entry of viral vectors into target cells expressing the above receptors.

[0007] Currently, vectors based on lentiviral backbones and VSVG envelope proteins have been widely used in clinical treatment. For example, in chimeric antigen receptor T-cell therapy (CAR-T), CAR-T is prepared by infecting T cells with lentiviruses carrying VSVG to encapsulate the CAR molecule gene.

[0008] Lentiviral vectors are a type of viral vector modified from the human immunodeficiency virus (HIV). They are a type of retrovirus with an RNA genome from which toxic genes have been deleted and replaced with an exogenous target gene, making them pseudotyped viruses. Reverse transcriptase is used to integrate exogenous genes into the genome for stable expression, allowing them to infect both dividing and non-dividing cells. The original HIV virus carries the gp120 and gp41 complex, which recognize CD4 molecules and facilitate HIV infection. Modified lentiviral vectors do not express gp120 and gp41, but instead express the VSVG envelope protein. Because VSVG can target and recognize the LDL-R, which is ubiquitously expressed, lentiviruses carrying VSVG can infect a wide range of cell types, including T cells, hepatocytes, cardiomyocytes, neurons, endothelial cells, stem cells, and various tumor cells.

[0009] The advantages of lentiviral vectors are: (1) long expression time: lentivirus can integrate foreign genes into the host cell genome, achieving long-term stable gene expression without loss during cell division and generation. It is the first choice for cell experiments and is often used to construct stable transgenic lines; (2) high safety: no pathogenicity has been found, and lentiviral vectors are used to transform T cells for CAR-T cell therapy; (3) low immunogenicity: direct injection into living tissue is unlikely to cause an immune response, making it suitable for animal experiments. Its disadvantages are that LDL-R is widely expressed in many types, so its targeting is not strong; and many cells are difficult to infect with lentiviruses, such as resting hematopoietic stem cells, resting T cells, NK cells, and B cells.

[0010] Currently, there are reports on the development of targeted lentiviruses. For example: 1. CD3 / CD28 antibody-based targeted lentiviruses. By expressing CD3 and CD28 antibodies or other T cell-activating antibodies with transmembrane sequences on the lentiviral surface, the viral particles can activate and infect T cells. This approach effectively enables lentiviral vectors to infect T cells in vivo. Although the presence of CD3 antibodies enhances the virus's targeting, the presence of wild-type envelope proteins such as VSVG or Cocal virus allows the virus to infect other cell types, further enhancing its targeting. 2. Fusogen-based targeted viruses. Unlike envelope proteins such as VSVG and BaEV, which combine receptor recognition and envelope fusion, the envelope glycoproteins of measles virus (MV) and Nipah virus (NiV) are composed of H / G and F proteins. The H / G proteins are responsible for receptor recognition. After H recognizes the receptor, the F protein (fusogen) undergoes a conformational change to mediate fusion of the viral envelope and the cell membrane. By linking the H / G protein to an antibody, such as a CD4 or CD8 antibody, and thus delivering the antibody to the extracellular space, the virus can be mediated to specifically recognize CD4+ or CD8+ T cells. Since adding antibodies to the H / G protein increases the steric hindrance of the H / G protein, the conformationally modified F protein may not be able to reach the cell membrane of the target cell, making it difficult to mediate fusion of the virus with the envelope.

[0011] In addition to lentiviruses, AAV vectors are widely used to deliver gene therapy drugs. Adeno-associated virus (AAV) is a single-stranded DNA virus, and the current scientific consensus is that it does not cause any human disease. Recombinant adeno-associated virus (rAAV), used as a gene therapy vector, carries a protein capsid that is nearly identical to wild-type AAV. However, the portion of the genome within the capsid that encodes viral proteins has been completely deleted and replaced with the therapeutic transgene. The only portion of the AAV genome that remains is the ITRs, which guide genome replication and viral vector assembly. Currently, most AAV clinical trials are focused on four major organs / tissues: the eye, liver, muscle, and central nervous system. Intravenous gene therapy targeting the liver has shown promise in addressing metabolic and hematologic disorders. Parkinson's disease, hemophilia A, and hemophilia B are the three indications with the most clinical trials.

[0012] The advantages of AAV vectors are: (1) non-pathogenicity, as the vector is derived from a non-pathogenic virus; (2) small particles that can penetrate tissues; and (3) easy preparation and extremely high packaging titers. At the same time, although different serotypes of AAV vectors have different infection preferences, their targeting is still insufficient.

[0013] Other carriers, including LNPs and ADC drug delivery systems, have also been widely used in clinical practice.

[0014] Messenger RNA (mRNA) encapsulated in LNPs is used to prepare COVID-19 vaccines, and extensive research has been conducted in the field of tumor vaccines. Because the lipid bilayer lacks targeting properties, it is difficult to target and infect specific cells. By linking CD5 antibodies to LNPs, mRNA expressing CAR-T molecules can be delivered to T cells for targeted delivery, thereby preparing CAR-T. However, since LNPs are naturally easily phagocytosed by dendritic cells (DCs) and macrophages, and are easily enriched in the liver; at the same time, after LNP administration, the biomolecular corona formed on the LNP contains hundreds of biomolecules, such as high-density lipoprotein HDL, which will affect its targeting. Therefore, even with targeted modified antibodies, its targeting is still not strong.

[0015] ADC drugs connect antibodies to cell-specific endocytic receptors and drugs through a hinge (linker). The linker is stable in the blood, so the drug is not released during intravenous infusion. After the antibody binds to the cell-specific endocytic receptor, it is encapsulated by the lysosome, at which point the linker is degraded by enzymes in the lysosome, thereby releasing the drug. The drug will not be degraded by the lysosome and does not need to rely on lysosomal escape to be effective. ADC drugs have strong targeting, but based on existing ADC delivery methods, it is difficult to deliver other drugs besides small molecule compound toxins to cells, especially biomacromolecule drugs. This is because biomacromolecules such as proteins and nucleic acids are easily degraded by lysosomes.

[0016] In summary, existing delivery vectors generally have difficulty in achieving targeted delivery to specific cells, especially in delivering biological macromolecules to specific cells and achieving their functions. Summary of the Invention

[0017] The first aspect of the present invention provides a targeting vector, which comprises: a first molecule that binds to the endocytic receptor of the target cell and a second molecule that promotes the release of the substance carried by the targeting vector into the cytoplasm; when the targeting vector is a viral vector, the first molecule is not part of the viral envelope protein.

[0018] Targeting vectors come in various forms and can be enveloped or non-enveloped vectors. Enveloped vectors include retroviral vectors, lentiviral vectors, and lipid nanoparticles; non-enveloped vectors include adenoviruses, adeno-associated viruses, and virus-like particles.

[0019] Endocytic receptors are membrane proteins expressed on the cell surface that can induce endocytosis (also known as pinocytosis) upon binding to antibodies, ligands, or specific substances. Examples include CD7, CD5, HER2, and mesothelin. Endocytic receptors typically contain domains such as YXXPhi, [D / E]XXXL[L / I], and FXNPXY. Furthermore, some membrane proteins, such as CD8, lack endocytic receptor domains and are generally considered to have weak endocytic abilities. Therefore, CD8 molecules with randomized sequences chimeric at the end of their intracellular sequences are often used for screening of endocytic domains.

[0020] Different cells express different, specific endocytic receptors. The first molecule can be designed according to the endocytic receptors of the cells to be targeted. Through antibodies or ligands (part of the first molecule) that specifically bind to the endocytic receptors, the vector is internalized by the target cells, thereby making the vector capable of infecting specific cells but not other cells.

[0021] After the first molecule binds to an endocytic receptor, it triggers receptor-mediated endocytosis. Receptor-mediated endocytosis is the process by which cells specifically take up extracellular proteins or other compounds through receptors on the cell surface. Cell surface receptors are highly specific and bind to their corresponding ligands to form complexes. This complex then indents the plasma membrane, forming coated pits. These pits then detach from the plasma membrane to form coated vesicles, which then take extracellular material into the cell. After entering the cell, the coated vesicles shed their outer coating and bind to smaller vesicles within the endosomal compartment to form larger endosomes or endosomes.

[0022] The role of the second molecule is to facilitate the endosomal or lysosomal escape of the targeting vector after it is internalized by the target cell, allowing it to enter the cytoplasm. If the targeting vector fails to escape endosomally or lysosomally after internalization, it will ultimately be degraded by the lysosomes. Therefore, the ability of the vector to escape endosomally or lysosomally is required for the effective delivery of the vector's payload into the cytoplasm.

[0023] Common ways of endosome / lysosome escape include: (1) destroying endosomes / lysosomes, such as polyethyleneimine, which causes the endosome / lysosome to swell and rupture through the "proton sponge" effect, allowing the carried substances to escape and enter the cytoplasm; (2) using the positive charge on the surface of the carrier to interact with the negatively charged lysosomal membrane to reduce the stability of the lysosomal membrane; (3) achieving escape through membrane fusion between the carrier and the endosome / lysosome, such as modifying the expression of the fusion peptide GALA in the vector, or the vector carries a viral envelope protein with membrane fusion ability, such as VSVG or its variants.

[0024] In one embodiment, the second molecule is a viral envelope protein and / or a non-viral envelope protein.

[0025] In a specific embodiment, the viral envelope protein is selected from at least one of the following: vesicular stomatitis virus envelope glycoprotein VSVG and its variants, baboon endogenous retrovirus envelope glycoprotein BaEV and its variants, feline endogenous retrovirus envelope glycoprotein RD114 and its variants, gibbon ape leukemia virus envelope glycoprotein GALV and its variants; the non-viral envelope protein is selected from at least one of VP1 and its variants of adeno-associated virus AAV, VP2 and its variants of adeno-associated virus AAV and polyethyleneimine.

[0026] Viral envelope proteins, such as VSVG, can promote the fusion of the viral envelope with the endosomal / lysosomal membrane in endosomes / lysosomes, thereby facilitating the release of the vector-carrying substances. Non-viral envelope proteins, such as VP1 and VP2 of AAV, can undergo conformational changes under acidic conditions and form pores in the endosomes / lysosomes, thereby releasing the vector-carrying substances. Polyethyleneimine can exert a proton sponge effect in endosomes / lysosomes. That is, when the pH in the lysosome decreases, PEI can capture a large number of protons and cause an influx of chloride ions, resulting in an increase in the osmotic pressure in the endosomes / lysosomes. Finally, the endosomes / lysosomes rupture, thereby facilitating the release of the vector-carrying substances.

[0027] A variant refers to a mutant having at least 75% identity to the amino acid sequence of a non-mutant (wild type), and "at least 75% identity" refers to 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% identical to the amino acid sequence of a non-mutant (wild type).

[0028] Because VSVG's receptor, LDL-R, is widely expressed in activated T cells, hepatocytes, cardiomyocytes, endothelial cells, stem cells, and other cell types, as well as various tumor cells, pseudotyped lentiviruses carrying VSVG or its variants can infect a variety of cell types. Although VSVG-based pseudotyped viruses have broad-spectrum infection capabilities, many cells, such as NK cells and resting T cells, do not express or express low levels of VSVG's receptor, LDL-R. In these cases, VSVG-based pseudotyped viruses are difficult to infect. Furthermore, because LDL-R is widely expressed in a variety of cell types, VSVG-based pseudotyped viruses lack targeting. For example, if multiple cell types express LDL-R, it is difficult for lentiviruses to precisely transfect only one of them.

[0029] This approach utilizes the endosomal / lysosomal escape ability of lentiviruses, modifies them, and connects a first molecule to the lentivirus. The resulting targeting vector can be flexibly applied to different scenarios targeting different cells. By designing the first molecule based on the cells to be targeted, the scope of application is greatly broadened, and the accuracy of targeting is also improved.

[0030] In a specific embodiment, the second molecule is selected from VSVG and variants thereof.

[0031] In one embodiment, the first molecule comprises a transmembrane peptide, an antibody or a ligand that binds to an endocytic receptor on a target cell. 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 the CD8 hinge region is commonly used.

[0032] The amino acid sequence of the first molecule is not limited unless it affects its function. For example, when the first molecule includes a CD33 antibody, the first molecule can 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.

[0033] Endocytic receptors of different target cells are different. 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, Collagen IV, Periostin, CEACAM, c-MET, TDGF1, IGF1R, Mesothelin, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, glycosphingolipid, TfR, Gan glioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, ASGPR.

[0034] In one specific 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. When the target cell does not express a receptor for the viral envelope protein, the virus can infect the target cell through endocytosis mediated by the first molecule.

[0035] In a specific embodiment, the transmembrane protein is selected from CD7 antibody, CD19 antibody, CD33 antibody, ASGRP antibody or ligand, Mesothelin antibody, HER2 antibody; the transmembrane protein can also be an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5% identical protein; the transmembrane protein may also be a protein having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical amino acid sequence to at least one of the CD8 signal peptide, Gemtuzumab light chain (VL), GS linker, Gemtuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the CD33 antibody; the transmembrane protein may also be a protein having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical amino acid sequence to at least one of the CD8 signal peptide, Gemtuzumab light chain (VL), GS linker, Gemtuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the CD19 antibody. The amino acid sequence of at least one of the peptide (linker), FMC-63 chain (VL), CD8 hinge region, and CD8 transmembrane region is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to that of at least one of the ASGRP antibody CD8 signal peptide, ASGPR light chain B11 (VL), GS connecting peptide (linker), ASGPR heavy chain (VH), CD8 hinge region, and CD8 transmembrane region. 0%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical protein; the transmembrane protein can also be a protein having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical amino acid sequence to at least one of the following: CD8 signal peptide, Pertuzumab light chain (VL), GS linker, Pertuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of HER2 antibody.5% identical protein; the transmembrane protein may also be a protein having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical amino acid sequence to at least one of the CD8 signal peptide, PE38 heavy chain (VH), GS linker, PE38 light chain (VL), CD8 hinge region, and CD8 transmembrane region of the MESOTHELIN antibody.

[0036] In one specific approach, a mutation in the viral envelope protein weakens its receptor recognition ability, such as reducing or eliminating its ability to bind to LDL-R, leaving it with only the ability to escape from the endosomal or lysosomal compartments, thus completely relying on the first molecule for its targeting. By carrying this mutated envelope protein, the targeting ability of the vector is further enhanced, allowing it to infect only specific target cells.

[0037] In a specific scheme, the first molecule contains an antibody or a ligand. After the antibody binds to the corresponding antigen (endocytic receptor) on the target cell, endocytosis occurs. The second molecule uses a mutated VSVG to destroy the binding ability of VSVG to LDL-R, but at the same time does not affect the lysosomal escape ability of VSVG. The mutation is an insertion, deletion or replacement. For example, the envelope glycoprotein VSVG of the Indiana strain of vesicular stomatitis virus contains one or more combinations of the following site mutations: H8 mutation, N9 mutation, Q10 mutation, K47 mutation, K50 mutation, A51 mutation , 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 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353.

[0038] Furthermore, VSVG comprises one or more combinations of the following site mutations: replacement of H8, replacement of N9, replacement of Q10, replacement of K47, deletion of K47, replacement of K50, replacement of A51, replacement of S183, replacement of S179, replacement of N180, replacement of I182, replacement of M184, replacement of Y209, replacement of I347, replacement of T350, replacement of T352, replacement of E353, replacement of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353.

[0039] Furthermore, K47 and / or R354 of the amino acid sequence of VSVG are mutated, for example, the 47th amino acid of VSVG is replaced by lysine K to glutamine Q, and / or the R354th amino acid is replaced by arginine R to glutamine Q, for example, the 47th amino acid of VSVG is deleted.

[0040] In a specific embodiment, the viral envelope protein is selected from the Indian strain envelope glycoprotein VSVG of vesicular stomatitis virus, 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.

[0041] In a specific embodiment, compared with the entire domain of 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 undergo mutations corresponding to the following sites: substitution / deletion of H8, substitution / deletion of N9, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of K50, substitution / deletion of A51, substitution / deletion of S183, substitution / deletion of S179, substitution / deletion of N180, substitution / deletion of I182, substitution / deletion of M184, substitution / deletion of Y209. Loss, substitution / deletion of I347, substitution / deletion of T350, substitution / deletion of T352, substitution / deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353.

[0042] Amino acid mutations include deletions, substitutions, and insertions. Most research and experiments focus on amino acid substitutions, while research on amino acid insertions and deletions remains largely neglected (Savino S, et al., Insertions and deletions in protein evolution and engineering. Biotechnol Adv. (2022)).

[0043] The substitution or deletion of amino acids at the same site can cause unpredictable and completely different effects.

[0044] The replacement of amino acid 47 at position 47 of the VSV-G extracellular domain from lysine K to glutamine Q (K47Q) and amino acid 354 at position 354 from arginine R to glutamine Q (R354Q) can both lead to a weakening or even loss of the ability of VSV-G to specifically bind to LDL-R; the result that a lentivirus using VSV-G containing the K47Q or R354Q mutation in the extracellular domain to construct an envelope glycoprotein and the viral envelope containing the aforementioned first molecule, i.e., a transmembrane protein, can still infect target cells specific to the targeting molecule does not necessarily predict that a lentivirus using VSV-G containing a K47 deletion or R354 deletion in the extracellular domain to construct an envelope glycoprotein and the viral envelope containing the aforementioned first molecule still has the ability to specifically infect target cells.

[0045] Lentiviruses constructed with VSV-G containing R354 deletion in the extracellular domain and containing membrane-type anti-CD7 antibodies were unable to effectively infect CD7. + However, lentivirus containing a membrane-type anti-CD7 antibody expressing VSV-G containing a K47 deletion in the extracellular domain can effectively infect CD7 cells. + This also further proves that the changes in protein function caused by amino acid deficiency are unpredictable.

[0046] Taking lentiviral T cell infection as an example, the lentiviral envelope expresses a mutant VSVG and CD7 antibody. When the CD7 antibody binds to the CD7 antigen on the T cell surface, it mediates the endocytosis of the lentivirus. The mutant VSVG then mediates lysosomal escape, completing the targeted infection of the lentivirus on the T cell. Furthermore, the lentivirus only infects T cells that express CD7 and has no ability to infect cells that do not express CD7.

[0047] The substances carried by the targeting vector are not limited and can be selected based on actual needs, such as small molecules, proteins, peptides, RNA, DNA, including chimeric antigen receptors (CARs) and TCRs. 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 that specifically bind to the 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., the CD8 transmembrane domain) and a signaling domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs) (e.g., CD3ζ). In some embodiments, the CAR comprises one or more costimulatory domains. The type of costimulatory domain is not limited to CARs. In fact, any costimulatory domain known in the art can be used, including but not limited to CD28, 4-1BB, DAP10, and DAP12.

[0048] Small molecule compounds refer to organic compounds with a molecular weight of less than 900 Daltons, especially small molecules that can be used as drugs.

[0049] In a specific embodiment, the target cells are lymphocytes, myeloid cells, hematopoietic stem / progenitor cells or non-blood cells, especially myeloid cells, hematopoietic stem / progenitor cells or non-blood cells, including normal cells and tumor cells.

[0050] In a specific embodiment, the endocytic receptor is an endocytic receptor of myeloid cells, an endocytic receptor of hematopoietic stem / progenitor cells, or an endocytic receptor of non-blood cells.

[0051] In a specific embodiment, the endocytic receptor is not CD80, TCR, BCR, CD19, CD20, or IL-13Rα.

[0052] In one embodiment, the endocytic receptor is not a lymphocyte-specific protein.

[0053] Lymphocyte-specific proteins refer to specific proteins of B cells, T cells or NK cells. 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 (CXCR4), 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 TCR (TCRγ, TCRδ), Vdeltal, Vdelta2, NKG2D (KLRK1, CD314), TCR (Va24-Jal8), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), VLA4.

[0054] 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.

[0055] NK cell-specific proteins include CD56, NKp46, CD16, KIR(s), NKG2 protein (NKG2D, KLRK1, CD314), KLRB1 (CD161), KLRD1 (CD94), IL2Rb (CD 122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R.

[0056] In another aspect of the present invention, a method for preparing a targeting vector comprises the following steps:

[0057] Design the first molecule based on the endocytic receptor of the target cell,

[0058] Select the second molecule,

[0059] The first molecule, the second molecule and the substance carried by the carrier are assembled to prepare a targeting carrier.

[0060] In the first case, by designing the first molecule, that is, modifying the virus surface with an antibody / ligand that can target and recognize a specific receptor, and selecting the wild-type viral envelope protein as the second molecule, if the target cell expresses the envelope protein receptor carried by the virus, then the presence of the first molecule can promote the infection of the target cell by the targeted vector.

[0061] In the second case, by designing the first molecule, that is, modifying the virus surface with antibodies / ligands that can target and recognize specific receptors, and selecting the wild-type viral envelope as the second molecule, if the target cell low-expresses or does not express the envelope protein receptor carried by the virus, then relying on the first molecule, the virus can still complete the infection of the target cell.

[0062] In the third case, by designing the first molecule, that is, modifying the virus surface with antibodies / ligands that can target and recognize specific receptors, and selecting a mutant viral envelope protein with limited receptor binding but the ability to escape from endosomes / lysosomes as the second molecule, regardless of whether the target cell expresses the envelope protein receptor carried by the virus, the virus can target and infect the target cell based on the first molecule, and the virus will not infect other cells, which greatly improves the accuracy of targeting.

[0063] Assembly methods of enveloped and non-enveloped vectors:

[0064] Assembly of enveloped vectors, taking VSVG as an example: a targeted expression vector can be constructed by mixing a plasmid expressing an antibody / ligand that binds to an endosomal receptor with a lentiviral packaging plasmid, such as psPAX2 and pMD.2G (VSVG or VSVG mutant), and a lentiviral expression vector.

[0065] The assembly of non-enveloped vectors, taking AAV as an example: the antibody / ligand that binds to the endocytic receptor is connected to the envelope protein of AAV through a transmembrane peptide segment to achieve the construction of the AAV targeting vector.

[0066] Envelope and non-enveloped vectors enter cells:

[0067] Enveloped vectors, such as pseudotyped lentiviral vectors based on VSVG, bind to LDL-R on the cell membrane and enter the cell through clathrin-mediated endocytosis. Following acidification of the endosomes formed by endocytosis, the conformation of VSVG changes, leading to fusion of the viral envelope with the endosomal membrane, allowing the virus to detach from the endosome / lysosome and enter the cell nucleus through the nuclear pore.

[0068] The non-enveloped vector, recombinant AAV virus particles, enter the cell through clathrin-mediated endocytosis by binding to the glycosylated receptors expressed on the host cell surface. After the endosome formed by endocytosis becomes acidified, the conformation of the VP1 / VP2 part of the viral capsid changes, causing the virus to detach from the endosome and enter the cell nucleus through the nuclear pore.

[0069] In another aspect of the present invention, the targeting vector can be used for drug or vaccine delivery, especially for the delivery of small molecule compounds, proteins, polypeptides, RNA or DNA.

[0070] Another aspect of the present invention provides a method for introducing a substance into a cell, the method comprising: contacting the cell with a targeting vector.

[0071] In one embodiment, the cell is a mammalian cell.

[0072] In one embodiment, the cell is a normal cell or a cancer cell.

[0073] In a specific embodiment, the cell is a T cell, a NK cell, a B cell, a macrophage, a granulocyte, a dendritic cell, a hematopoietic stem cell, a hepatocyte, a pancreatic islet cell, a nerve cell, or a muscle cell.

[0074] In a specific embodiment, the contact is performed in vivo or in vitro, for example, the targeted vector can be administered intravenously, intraperitoneally, intratumorally, intrabone or intranodally to allow the targeted vector to enter the body and contact the target cells; or the target virus can be directly infected with the target cells in vitro.

[0075] The term "mammal" refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0076] The term "viral envelope protein" refers to naturally occurring viral envelope proteins, such as VSV-G, BaEV, and RD114. Viral envelope proteins play an important role in viral packaging and infection of host cells.

[0077] Another aspect of the present invention provides a composition comprising a targeting vector; the composition can be used as a drug; the composition can be used to prepare drugs for gene therapy, immunotherapy, cell therapy, treatment of gene defect diseases, treatment of autoimmune diseases, treatment of infectious diseases, and treatment of cancer; the cancers include blood cancers and solid cancers.

[0078] Another aspect of the present invention is a method for treating a disease in a subject, comprising administering a therapeutically effective amount of a targeted vector or composition to the subject.

[0079] The routes of administration / administration of the pharmaceutical composition are conventional in the art, such as oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered by sustained release system or by implantation device.

[0080] "Treatment" refers to treating a subject with the treatments described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). The treatment methods that effectively treat a patient can vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the treatment to elicit an anti-cancer response in the subject.

[0081] Those skilled in the art will appreciate that appropriate dosage levels for treatment will vary depending, in part, on the molecule being delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and / or status (age and general health). In certain embodiments, the clinician can titrate the dosage and vary the route of administration to obtain the optimal therapeutic effect.

[0082] This solution utilizes a combination of endocytosis and endosomal / lysosomal escape to modify the vector, making it carry a first molecule that can bind to the endocytic receptor of the target cell, thereby obtaining a targeted vector with the following beneficial effects:

[0083] Based on the efficient endocytic receptors specific to different cell types, targeted vectors can be developed quickly and specifically for different cell types without the need for blind screening or trial-and-error verification;

[0084] Designing the first molecule based on the endocytic receptors of the cells to be targeted can target different types of cells, especially cells that do not express or low-express widely used viral envelope protein receptors (such as LDL-R), and should have a wide range;

[0085] After reasonable mutation of the vector, infection of unwanted targeted cells can be avoided, and the accuracy is greatly improved.

[0086] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety, just as if each individual publication, document, or patent were not specifically and individually indicated as being incorporated by reference in its entirety. In the event of a conflict, the present application (including any definitions herein) will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0087] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] FIG1 is a schematic diagram of a targeting vector;

[0089] FIG2 is a schematic diagram of the pGClenti-GFP lentiviral vector backbone;

[0090] FIG3 is a flow cytometric graph of virus-infected peripheral blood NK cells in Example 1;

[0091] FIG4 is a schematic diagram of the VSVG mutant of Example 2;

[0092] FIG5 is a graph showing detection of capsid protein P24 of 15 groups of lentiviruses using different VSVG mutants to construct envelope glycoproteins;

[0093] FIG6 is a graph showing titer detection of 15 groups of lentiviruses using different VSVG mutants to construct envelope glycoproteins;

[0094] Figure 7 is a diagram of viral infection of CD7 in Example 3 + Flow cytometry of cells;

[0095] Figure 8 is a flow cytometry graph showing the infection of Jurkat cells, Nalm6 cells and THP1 cells with the targeted lentivirus A33-VSV-G-1;

[0096] Figure 9 is a flow cytometry graph showing the infection of Jurkat cells and THP1 cells with the targeted lentivirus A33-VSV-G-2;

[0097] Figure 10 is a flow cytometry graph showing the infection of Jurkat cells and Nalm6 cells with the targeted lentivirus A19-VSV-G-2;

[0098] Figure 11 is a graph showing titer detection of targeted lentivirus A19-VSV-G-1 and A19-VSV-G-2;

[0099] Figure 12 is a flow cytometry graph showing the infection of Jurkat cells, HuH-7 cells and Hep-G2 cells with the targeted lentivirus A-ASGPR-VSV-G-2;

[0100] Figure 13 is a flow cytometry graph showing the infection of Jurkat cells and MCF7 cells with the targeted lentivirus A-HER-2-VSV-G-2;

[0101] Figure 14 is a flow cytometry graph showing the infection of Jurkat cells and Jurkat-MSN cells with the targeted lentivirus A-Jurkat-MSN-VSV-G-2;

[0102] Figure 15 is a flow cytometry graph showing the infection of Jurkat cells, Nalm6 cells and PBMCs with the targeted lentivirus A8-VSV-G-1;

[0103] Figure 16 is a flow cytometric graph showing infection of Jurkat cells and PBMCs with the targeted lentivirus A8-VSV-G-2;

[0104] Figure 17 is a flow cytometric graph showing Jurkat cells infected with lentivirus dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, respectively;

[0105] FIG18 is a flow cytometer graph showing that Nalm6 cells were infected with lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, respectively. DETAILED DESCRIPTION

[0106] Example 1

[0107] Constructing NK-targeting lentivirus using wild-type VSVG

[0108] Because NK cells have low expression of the VSVG receptor, LDL-R, ordinary VSVG lentiviruses have difficulty infecting NK cells. Therefore, the inventors constructed a membrane-expressed CD7 antibody on the lentiviral envelope, allowing it to bind to CD7 on NK cells and induce endocytosis of the lentiviral. VSVG then mediates the fusion of the viral envelope and the endosomal / lysosomal membrane, resulting in lysosomal escape, allowing the vector-loaded GFP to enter the cell for expression. The structure of the lentiviral targeting vector is shown in Figure 1, where 1 is the first molecule, CD7 antibody, which can target the endocytic receptor, and 2 is the second molecule, VSVG, which mediates endosomal / lysosomal escape.

[0109] 1. Design of membrane-expressed CD7 antibodies

[0110] 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, a TH-69 light chain (VL), a CD8 hinge region, and a CD8 transmembrane region. The amino acid sequence of the CD8 signal peptide is SEQ ID NO: 1:

[0111] MALPVTALLLPLALLLHAARP

[0112] The amino acid sequence of the TH-69 heavy chain (VH) is SEQ ID NO: 2:

[0113] EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS

[0114] The amino acid sequence of the GS linker peptide is SEQ ID NO: 3:

[0115] GGGGSGGGGSGGGGS

[0116] The amino acid sequence of the TH-69 light chain (VL) is SEQ ID NO: 4:

[0117] AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR

[0118] The amino acid sequence of the CD8 hinge region is SEQ ID NO: 5:

[0119] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0120] The amino acid sequence of the CD8 transmembrane region is SEQ ID NO: 6:

[0121] IYIWAPLAGTCGVLLLSLVITLYC

[0122] 2. Construction of wild-type VSVG

[0123] The wild-type extracellular domain of VSVG comprises the amino acid sequence shown in SEQ ID NO:7:

[0124] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0125] 3. Encapsulated lentivirus

[0126] A plasmid carrying a membrane-expressing CD7 antibody, psPAX2, and pMD2.G (VSVG wild-type) packaging plasmids were used to encapsulate a lentivirus expressing pGClenti-GFP (GFP fluorescent protein) (the backbone is shown in Figure 2). 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 the lentivirus that targets NK cells. The lentivirus was then added to the NK cell culture system to infect NK cells. The results are shown in Figure 3. The left figure shows GFP expression in NK cells infected with the VSVG lentivirus; the right figure shows GFP expression in NK cells infected with the VSVG lentivirus carrying the membrane-bound CD7 antibody. While standard VSVG lentiviruses have difficulty infecting NK cells, the VSVG lentivirus carrying the membrane-bound CD7 antibody effectively targets NK target cells because NK cells do not express LDL-R but highly express CD7.

[0127] Example 2

[0128] Screening for VSVG mutants that lack receptor binding ability but retain endosomal / lysosomal escape ability

[0129] Based on the binding site of VSVG to LDL-R, the inventors designed a series of VSVG mutants, as shown in Figure 4, where Mut represents mutant and Δ represents base deletion, to test their ability to bind to the receptor and escape from endosomes / lysosomes.

[0130] The 15 groups of VSV-G mutants shown in FIG4 were used to construct viral envelope proteins. Referring to the method for packaging lentiviruses described in Example 1, 15 groups of lentiviruses containing membrane-type lentiviruses expressing anti-CD7 antibodies were packaged.

[0131] The capsid protein P24 of the 15 groups of packaged lentiviruses was tested, and the results are shown in FIG5 . The method for detecting the capsid protein P24 is well known to those skilled in the art.

[0132] The 15 groups of lentivirus obtained by packaging were used to infect CD7 + The infection ability of each group of lentiviruses was tested in Jurkat cells, and the results are shown in Figure 6.

[0133] As shown in Figure 5 , the lentiviral capsid protein P24 was detected in all 15 groups of packaged lentiviruses, proving that each group successfully packaged the lentivirus; however, as shown in Figure 6 , there were significant differences in the biological activity and titer of the 15 groups of packaged lentiviruses.

[0134] As can be seen from Figure 6, compared with lentiviruses using other VSV-G mutants to construct envelope glycoproteins, the titer of the lentivirus using the K47 deleted VSV-G mutant to construct viral envelope glycoproteins is significantly improved, which is about twice the titer of the lentiviruses using the R354Q mutant VSV-G mutant and the K47Q mutant VSV-G mutant to construct viral envelope glycoproteins; while the titer of the lentivirus using the R354 deleted VSV-G mutant to construct viral envelope glycoprotein is close to 0, and has almost no infection ability.

[0135] Example 3

[0136] Construction of lentivirus targeting Jurkat cells using mutant VSVG

[0137] The mutant VSVG is used to retain its ability to escape lysosomes but lose its ability to bind to LDL-R, allowing it to specifically target cells expressing CD7. The lentivirus cannot infect cells that do not express CD7 but express LDL-R.

[0138] 1. Design of CD7 Antibody

[0139] Same as Example 1.

[0140] 2. Construction of mutant VSVG

[0141] The extracellular domain of the VSVG mutant (amino acid mutated to Q at position 354) comprises the amino acids shown in SEQ ID NO: 8:

[0142] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEQELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0143] 3. Encapsulated lentivirus

[0144] A targeted lentivirus expressing pGClenti-GFP (GFP fluorescent protein) is encapsulated with a plasmid carrying a membrane-expressing CD7 antibody, psPAX2, and a mutant packaging plasmid for VSVG. Specifically, the four plasmids are mixed and transfected into 293T cells using PEI. After 48 hours, the culture supernatant is collected and centrifuged to obtain a lentivirus that targets CD7+ cells, including Jurkat cells, CD7+ NK cells, and CD7+ T cells. Adding the lentivirus to the culture system of the above cells infects the corresponding target cells.

[0145] The results are shown in Figure 7. Raji cells that do not express CD7 and Jurkat cells that express CD7 were infected with VSVG lentivirus and targeted lentivirus, respectively. 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 is Raji cells, which do not express CD7, and the experimental group is CD7+ Jurkat cells. The non-targeted lentiviral vector has the ability to infect both Raji cells and Jurkat cells, while the targeted lentiviral vector only has the ability to infect Jurkat cells and has no ability to infect Raji cells. The CD7-targeted lentivirus in this example cannot effectively infect Raji cells, but can only effectively infect Jurkat cells. This is because the mutant VSVG used in the targeted lentivirus has no ability to infect cells. Its targeting depends on the CD7 antibody and can only target cells expressing CD7. The wild-type VSVG carried by the non-targeted lentivirus can bind to cells expressing LDL-R, regardless of whether the cell expresses CD7.

[0146] Example 4

[0147] Construction of lentivirus targeting CD33+ cells

[0148] 1. Design of membrane-expressed CD33 antibodies

[0149] The membrane-expressed CD33 antibody sequence includes, from 5' to 3' end, the CD8 signal peptide, Gemtuzumab light chain (VL), GS linker, Gemtuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region.

[0150] The amino acid sequence of the gemtuzumab light chain (VL) is SEQ ID NO: 9:

[0151] DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVE VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0152] The amino acid sequence of the gemtuzumab heavy chain (VH) is SEQ ID NO: 10:

[0153] EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0154] 2. Referring to Example 3, construct mutant VSV-G

[0155] Referring to Example 3, a mutant VSV-G was constructed. The extracellular domain of the VSV-G mutant comprised the amino acid sequence shown in SEQ ID NO: 8 (the arginine at position 354 was mutated to glutamine Q, mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO: 21 (the lysine K at position 47 was deleted, mutant VSV-G-2).

[0156] 3. Encapsulated lentivirus

[0157] Referring to Example 3, the targeted lentivirus A33-VSV-G-1 and A33-VSV-G-2 were packaged and used to infect CD33 cells. + cells and CD33 - cell.

[0158] Take 0.5×10 6 Jurkat cells, Nalm6 cells and THP1 cells were resuspended in 200 μL of culture medium, which included 1640 (brand: Elgbio, product number: EH80809) medium and 10% FBS (brand: EXCELL, product number: FSP500). At an MOI of 1, the CD33-targeting +The cell-derived lentivirus A33-VSV-G-1 was mixed and placed in a 37°C incubator for culture. On Day 2, the expression of GFP in Jurkat cells, Nalm6 cells, and THP1 cells was detected. The results are shown in Figure 8.

[0159] As shown in Figure 8, the targeted lentivirus A33-VSV-G-1 cannot effectively infect Jurkat cells and Nalm6 cells that express LDL-R but do not express CD33, but can effectively infect THP1 cells that express CD33.

[0160] Referring to the above-mentioned method of infecting Jurkat cells and THP1 cells with the targeted lentivirus A33-VSV-G-1, 1×10 5 Jurkat cells and THP1 cells were cultured, and the targeted lentivirus A33-VSV-G-2 was added to the Jurkat cell culture system and the THP1 cell culture system, respectively, at an MOI of 1. The expression of GFP in each culture system was detected on Day 2. The results are shown in FIG9 .

[0161] As shown in Figure 9, the targeted lentivirus A33-VSV-G-2 cannot effectively infect Jurkat cells that express LDL-R but do not express CD33, but can effectively infect THP1 cells that express CD33.

[0162] In this example, the mutant VSV-G-1 or VSV-G-2 is used to construct the targeted lentivirus A33-VSV-G-1 and A33-VSV-G-2. The viral envelope contains membrane-expressed anti-CD33 antibodies that can specifically bind to CD33. + The cell surface endocytic receptor CD33 enters and infects CD33 through endocytosis + cells; at the same time, because mutant VSV-G-1 or VSV-G-2 is used to construct its viral envelope glycoprotein, the ability of the targeted lentivirus A33-VSV-G-1 and A33-VSV-G-2 to specifically bind to LDL-R is weakened or lost, and the cells expressing LDL-R cannot be effectively infected, which effectively improves the infection rate of the targeted lentivirus A33-VSV-G-1 and A33-VSV-G-2 with CD33 + Cell targeting.

[0163] Flow cytometry antibody used for CD33 detection: Trade name: APC-CD33, Brand: Biolegend, Catalog number: #366606.

[0164] Example 5

[0165] Constructing a CD19-targeted + Lentivirus

[0166] 1. Design membrane to express CD19 antibody

[0167] The membrane-expressed CD19 antibody sequence includes, from 5' to 3' end, the CD8 signal peptide, FMC-63 heavy chain (VH), GS linker, FMC-63 light chain (VL), CD8 hinge region, and CD8 transmembrane region.

[0168] The amino acid sequence of the FMC-63 heavy chain (VH) is shown in SEQ ID NO: 11;

[0169] The amino acid sequence of the FMC-63 light chain (VL) is shown in SEQ ID NO: 12;

[0170] 2. Referring to Example 3, construct mutant VSV-G

[0171] Referring to Example 3, a mutant VSV-G was constructed. The extracellular domain of the VSV-G mutant comprised the amino acid sequence shown in SEQ ID NO: 8 (the arginine at position 354 was mutated to glutamine Q, mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO: 21 (the lysine K at position 47 was deleted, mutant VSV-G-2).

[0172] 3. Encapsulated lentivirus

[0173] Referring to Example 3, the mutant VSV-G-1 and mutant VSV-G-2 were used to construct targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2.

[0174] Take 1×10 5 CD19 - Jurkat cells and CD19 + Nalm6 cells were resuspended in 200 μL of culture medium, which included 1640 (Brand: Elgbio, Catalog No.: EH80809) medium and 10% FBS (Brand: EXCELL Catalog No.: FSP500). At an MOI of 1, targeting CD19 was added to each cell culture system. + The cell-targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 were mixed and cultured in a 37°C incubator. The expression of GFP in the Jurkat cells and Nalm6 cells was detected on Day 2. The results of infection of Jurkat cells and Nalm6 cells with the targeted lentivirus A19-VSV-G-2 are shown in Figure 10 ; the titer detection results of the targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 are shown in Figure 11 .

[0175] As shown in Figure 10, the targeted lentivirus A19-VSV-G-2 cannot effectively infect Jurkat cells that do not express CD19, but can effectively infect Nalm6 cells that express CD19.

[0176] The targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 specifically bind to the endocytic receptor CD19 on the surface of Nalm6 cells through the anti-CD19 antibody contained in their viral envelope, and then enter and infect CD19 through endocytosis. + Nalm6 cells.

[0177] As can be seen from Figure 11, compared with the targeted lentivirus A19-VSV-G-1 whose envelope glycoprotein is constructed using the mutant VSV-G-1 (R354Q mutation), the titer of the targeted lentivirus A19-VSV-G-2 whose envelope glycoprotein is constructed using the mutant VSV-G-2 (K47 deletion) is significantly improved.

[0178] Flow cytometry antibody used for CD19 detection: Trade name: APC-CD19, brand: Sianchi, item number: #S0098.

[0179] Example 6

[0180] Construct a lentivirus targeting asialoglycoprotein receptor-positive (ASGPR+) hepatocytes.

[0181] Targeting the asialoglycoprotein receptor (ASGPR) can be done with N-acetylgalactosamine (GalNAc) or an ASGPR antibody. GalNAc can be added to the surface of the lentiviral envelope through modification, or an ASGPR antibody can be expressed on the membrane to generate a lentivirus targeting hepatocytes.

[0182] 1. Design of membrane-expressed ASGPR antibodies

[0183] The membrane-expressed CD33 antibody sequence includes, from 5' to 3' end, the CD8 signal peptide, ASGPR light chain B11 (VL), GS linker, ASGPR heavy chain (VH), CD8 hinge region, and CD8 transmembrane region.

[0184] The amino acid sequence of the ASGPR antibody light chain (VL) is SEQ ID NO: 13:

[0185] DIVLTQPPSASGTPGQRVTISCTGSSSGIGNAYVSWYQQLPGKAPKLLIYKNGQRPSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKVTVL

[0186] The amino acid sequence of the ASGPR antibody heavy chain (VH) is SEQ ID NO: 14:

[0187] MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSAITTGGGSPNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRTAGYFDYWGQGALVTVSSGSA

[0188] 2. Referring to Example 3, construct mutant VSV-G

[0189] Referring to Example 3, a mutant VSV-G-2 was constructed, wherein the extracellular domain of the mutant VSV-G-2 comprises the amino acid sequence shown in SEQ ID NO: 21.

[0190] 3. Encapsulated lentivirus

[0191] Referring to Example 3, the mutant VSV-G-2 was used to construct a targeted lentivirus A-ASGPR-VSV-G-2.

[0192] Take 1×10 5 ASGPR - Jurkat cells and ASGPR + HuH-7 cells and Hep-G2 cells were resuspended in 200 μL of culture medium, which included DMEM medium and 10% FBS (DMEM, brand: Gibco, catalog number: #C12430500BT; FBS, brand: Yikesai, catalog number: # FSP500). At an MOI of 1, the target ASGPR was added to each cell culture system. + The cell-targeted lentivirus A-ASGPR-VSV-G-2 was mixed and placed in a 37° C. incubator for culture. On Day 2, the expression of GFP in Jurkat cells, HuH-7 cells, and Hep-G2 cells was detected. The results are shown in FIG12 .

[0193] As shown in Figure 12, the targeted lentivirus A-ASGPR-VSV-G-2 cannot effectively infect Jurkat cells that do not express ASGPR, but can effectively infect HuH-7 cells and Hep-G2 cells that express ASGPR.

[0194] The targeted lentivirus A-ASGPR-VSV-G-2 specifically binds to ASGPR through the anti-ASGPR antibody contained in its viral envelope. + The endocytic receptor ASGPR on the cell surface enters and infects ASGPR through endocytosis+ HuH-7 cells and Hep-G2 cells.

[0195] Flow cytometry antibody used for detection of ASGPR: Trade name: PE-ASGPR1, brand: BD, item number: #563655.

[0196] Example 7

[0197] Construction of lentivirus targeting HER2+ cells

[0198] 1. Design of membrane-expressed HER2 antibody

[0199] The membrane-expressed HER2+ antibody sequence includes, from 5' to 3' end, the CD8 signal peptide, Pertuzumab light chain (VL), GS linker, Pertuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region.

[0200] The amino acid sequence of the pertuzumab light chain (VL) is SEQ ID NO: 15:

[0201] DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0202] The amino acid sequence of the pertuzumab heavy chain (VH) is SEQ ID NO: 16:

[0203] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0204] 2. Referring to Example 3, construct mutant VSV-G

[0205] Referring to Example 3, a mutant VSV-G-2 was constructed, wherein the extracellular domain of the mutant VSV-G-2 comprises the amino acid sequence shown in SEQ ID NO: 21.

[0206] 3. Encapsulated lentivirus

[0207] Referring to Example 3, the mutant VSV-G-2 was used to construct a targeted lentivirus A-HER-2-VSV-G-2.

[0208] Take 1×10 5 HER-2 - Jurkat cells and HER-2 + The MCF7 cells were resuspended in 200 μL of culture medium, which included DMEM medium and 10% FBS (DMEM, brand: Gibco, catalog number: #C12430500BT; FBS, brand: Yikesai, catalog number: # FSP500). At an MOI of 1, the HER-2-targeting protein was added to the cell culture system of each group. + The cell-targeted lentivirus A-HER-2-VSV-G-2 was mixed evenly and placed in a 37° C. incubator for culture. On Day 2, the expression of GFP in Jurkat cells and MCF7 cells was detected. The results are shown in FIG13 .

[0209] As shown in FIG13 , the targeted lentivirus A-HER-2-VSV-G-2 cannot effectively infect Jurkat cells that do not express HER-2, but can effectively infect MCF7 cells that express HER-2.

[0210] The targeted lentivirus A-HER-2-VSV-G-2 specifically binds to the endocytic receptor HER-2 on the surface of MCF7 cells through the anti-HER-2 antibody contained in its viral envelope, and then enters and infects HER-2 through endocytosis. + MCF7 cells.

[0211] Flow cytometry antibody used for detection of HER-2: Trade name: APC-CD340 (HER-2), brand: Biolegend, catalog number: #324407.

[0212] Example 8

[0213] Construction of lentivirus targeting MESOTHELIN+ cells

[0214] 1. Design of membrane-expressed MESOTHELIN antibody

[0215] The membrane-expressed MESOTHELIN+ (MSN) antibody sequence includes, from 5' to 3' end, the CD8 signal peptide, PE38 heavy chain (VH), GS linker, PE38 light chain (VL), CD8 hinge region, and CD8 transmembrane region.

[0216] The amino acid sequence of the PE38 heavy chain (VH) is SEQ ID NO: 17:

[0217] MQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGV

[0218] The amino acid sequence of the PE38 light chain (VL) is SEQ ID NO: 18:

[0219] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIK

[0220] 2. Referring to Example 3, construct mutant VSV-G

[0221] Referring to Example 3, a mutant VSV-G-2 was constructed, wherein the extracellular domain of the mutant VSV-G-2 comprises the amino acid sequence shown in SEQ ID NO: 21.

[0222] 3. Encapsulated lentivirus

[0223] Referring to Example 3, the mutant VSV-G-2 was used to construct a targeted lentivirus A-MSN-VSV-G-2.

[0224] 4. Construction of Jurkat-MSN Overexpression Cell Line

[0225] A common lentivirus with wild-type VSV-G as envelope glycoprotein carrying the nucleic acid encoding MSN was used to infect the Jurkat cell line to construct a Jurkat-MSN cell line overexpressing MSN. The method for constructing the overexpression cell line is well known to those skilled in the art.

[0226] Take 1×10 5 MSN - Jurkat cells and MSNs + Jurkat-MSN cells were resuspended in 200 μL of culture medium, which included 1640+10% FBS (1640, brand: Elgbio, catalog number: #EH80809; FBS, brand: Yikesai, catalog number: #FSP500). At an MOI of 1, targeted MSNs were added to the cell culture system of each group. + The cell-targeted lentivirus A-MSN-VSV-G-2 was mixed and placed in a 37° C. incubator for culture. The expression of GFP in Jurkat cells and Jurkat-MSN cells was detected on Day 2. The results are shown in FIG14 .

[0227] As shown in Figure 14, the targeted lentivirus A-Jurkat-MSN-VSV-G-2 cannot effectively infect Jurkat cells that do not express MSN, but can effectively infect Jurkat-MSN cells that express MSN.

[0228] The targeted lentivirus A-MSN-VSV-G-2 specifically binds to the endocytic receptor MSN on the surface of Jurkat-MSN cells through the anti-MSN antibody contained in its viral envelope, and then enters and infects MSN through endocytosis. + Jurkat-MSN cells.

[0229] Flow cytometry antibody used for MSN detection: Trade name: APC-Mesothelin, brand: R&D, catalog number: #FAB32652A.

[0230] Example 9

[0231] Construction of a lentivirus targeting CD8+ cells based on an antibody against the non-endocytic receptor CD8

[0232] 1. Design of membrane-expressed CD8 antibodies

[0233] The membrane-expressed CD8+ antibody sequence includes, from 5' to 3' end, the CD8 signal peptide, CD8 antibody heavy chain (VH), GS linker, CD8 antibody light chain (VL), CD8 hinge region, and CD8 transmembrane region.

[0234] The amino acid sequence of the CD8 antibody heavy chain (VH) is SEQ ID NO: 19:

[0235] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS

[0236] The amino acid sequence of the CD8 antibody light chain (VL) is SEQ ID NO: 20:

[0237] DVQITQSPSSSLSASVGDRVTITTCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIK

[0238] 2. Referring to Example 3, construct mutant VSV-G

[0239] Referring to Example 3, a mutant VSV-G was constructed. The extracellular domain of the VSV-G mutant comprised the amino acid sequence shown in SEQ ID NO: 8 (amino acid at position 354 was mutated to Q, mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO: 21 (amino acid lysine K at position 47 was deleted, mutant VSV-G-2).

[0240] 3. Encapsulated lentivirus

[0241] 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 contained a membrane-type anti-CD8 antibody expression.

[0242] Take 1×10 5Jurkat cells, Nalm6 cells, and PBMCs were each resuspended in 200 µL of culture medium. The Jurkat and Nalm6 cells were resuspended in 1640 medium (Elgbio, Catalog No. EH80809) and 10% FBS (Excel, Catalog No. FSP500). PBMCs were resuspended in XVT medium (PRIME-XV T cell CDM, IRVINE (FUJIFILM), Catalog No. 91154), IL-7 (IL-7 Protein, Human, Recombinant, Sino Biological, Catalog No. 11821-HNAE) at a final concentration of 20 ng / mL, and IL-15 (IL-15 Protein, Human, Recombinant (His Tag), Sino Biological, Catalog No. 10360-H07E) at a final concentration of 20 ng / mL.

[0243] At an MOI of 1, the targeted lentivirus A8-VSV-G-1 was added to the Jukart cells, Nalm6 cells, and PBMCs cell culture systems, respectively. The mixture was evenly mixed and placed in a 37°C incubator for culture. On Day 2, the expression of GFP in the Jurkat cells, Nalm6 cells, and PBMCs was detected. The results are shown in FIG15 .

[0244] As shown in Figure 15, the targeted lentivirus A8-VSV-G-1 constructed using mutant VSV-G-1 cannot effectively infect Jurkat cells and Nalm6 cells that express LDL-R but do not express CD8, nor can it effectively infect PBMCs that express CD8.

[0245] Take 1×10 5 Jurkat cells and PBMCs were cultured in the same manner as described above. Referring to the method for mixing and culturing the targeted lentivirus A8-VSV-G-1 with each group of cells, the targeted lentivirus A8-VSV-G-2 was added to the Jurkat cell culture system and the PBMCs culture system, respectively, at an MOI of 1. The expression of GFP in the Jurkat cells and PBMCs was detected on Day 2. The results are shown in FIG16 .

[0246] As shown in FIG16 , the targeted lentivirus A8-VSV-G-2 cannot effectively infect Jurakt cells that express LDL-R but do not express CD8, nor can it effectively infect PBMCs that express CD8.

[0247] CD8 +After the cells were contacted with 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 was bound to CD8 in PBMCs. + The targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2 can not effectively enter and infect CD8. + cell.

[0248] The results showed that after contact between CD8+ cells and lentivirus, although the CD8 receptor can bind to the antibody, it cannot be effectively internalized. Lentiviral vectors based on membrane-expressed CD8 antibodies cannot effectively mediate infection of CD8+ cells. Therefore, receptors that lack efficient internalization are not suitable as the first molecule for constructing the targeting vector of the present invention.

[0249] Flow cytometry detection of CD8 used flow cytometry antibody: APC-CD8, brand: BD, product number: 566852.

[0250] Example 10

[0251] Comparison of infection efficiency of lentivirus containing K47 deletion or R354 deletion in the VSV-G extracellular domain.

[0252] Prepare an envelope plasmid carrying a nucleic acid encoding the membrane-expressing anti-CD7 antibody described in Example 1 and a nucleic acid encoding VSV-G with 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; the K47 deletion-A7 envelope plasmid is synthesized by conventional molecular cloning methods;

[0253] Referring to the packaging method for preparing lentivirus described in Example 3, lentivirus dK47-VSV-G-A7 was packaged and prepared;

[0254] Among the above four plasmids, the envelope plasmid containing a K47 deletion in the VSV-G extracellular domain was replaced with an envelope plasmid containing a R354 deletion, K47Q, or R354Q mutation in the VSV-G extracellular domain. Referring to the above method for packaging the lentiviral vector dK47-VSV-G-A7, lentivirus dR354-VSV-G-A7, sK47Q-VSV-G-A7, or sR354Q-VSV-G-A7 containing a R354 deletion, K47Q, or R354Q mutation in the VSV-G extracellular domain were packaged and prepared;

[0255] The VSV-G extracellular domain comprising a K47 deletion comprises the amino acid sequence shown in SEQ ID NO: 21;

[0256] The VSV-G extracellular domain comprising the R354 deletion comprises the amino acid sequence shown in SEQ ID NO: 22;

[0257] The VSV-G extracellular domain comprising the K47Q mutation comprises the amino acid sequence shown in SEQ ID NO: 23;

[0258] The VSV-G extracellular domain comprising the R354Q mutation comprises the amino acid sequence shown in SEQ ID NO: 8.

[0259] According to MOI=1, the above four lentiviruses were added into four groups of CD7 + Jurkat cells were mixed and infected at room temperature for 10 minutes. Then, 10 mL of DPBS buffer was added and mixed. The cells were then centrifuged at 500 g for 3 minutes. The supernatant was discarded and 1 mL of 1640 medium containing 10% FBS (brand: ELGBIO, product number: #EH80809; FBS serum: brand: EXCELL, product number: #FSP500) was added. The four groups of CD7 + The Jurkat cells were cultured in vitro in an incubator at 37°C and a CO2 concentration of 5%. The expression of GFP was detected on Day 2. The results are shown in FIG17 .

[0260] As shown in Figure 17, the lentivirus dK47-VSV-G-A7, sK47Q-VSV-G-A7 and sR354Q-VSV-G-A7 containing K47 deletion, R354Q or K47Q mutation in the VSV-G extracellular domain can effectively infect CD7 by specifically binding to the endocytic receptor CD7 expressed by Jurkat cells through endocytosis. + Jurkat cells; however, the lentivirus dR354-VSV-G-A7 containing the R354 deletion in the VSV-G extracellular domain was difficult to infect CD7 cells through endocytosis. + This indicates that after the R354 deletion of the VSV-G extracellular domain, the function of VSV-G has undergone an unknown and unpredictable change that is different from the K47 deletion; even if its viral envelope contains membrane-expressed anti-CD7 antibodies, the lentivirus dR354-VSV-G-A7 still loses the ability to infect CD7 + Cell capacity.

[0261] 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 into four groups of CD7 cells at an MOI of 1. - The Nalm6 cells were cultured in vitro in an incubator at 37°C and a CO2 concentration of 5%. The expression of GFP was detected on Day 2. The results are shown in FIG18 .

[0262] As shown in Figure 18, the lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7 and sR354Q-VSV-G-A7 were unable to infect CD7 - This indicates that the deletion of K47 in the extracellular domain of VSV-G does not affect the infection of CD7 by the lentivirus dK47-VSV-G-A7. + Cell targeting.

[0263] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in 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 based on the scope of protection 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; when the targeting vector is a viral vector, the first molecule is not part of the viral envelope protein.

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

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

4. The targeting vector according to claim 3, characterized in that The enveloped vector is selected from retroviral vectors, lentiviral vectors, and lipid nanoparticles; the non-enveloped vector is selected from adenovirus, adeno-associated virus, and virus-like particles.

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

6. The targeting vector according to claim 5, characterized in that The viral envelope protein is selected from at least one of the envelope glycoproteins VSVG and variants thereof of vesicular stomatitis virus, Cocal virus envelope glycoprotein and variants thereof, Maraba virus envelope glycoprotein and variants thereof, Morreton virus envelope glycoprotein and variants thereof, Alagoa virus envelope glycoprotein and variants thereof, New 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; the non-viral envelope protein is selected from at least one of VP1 and variants thereof of adeno-associated virus AAV, VP2 and variants thereof of adeno-associated virus AAV, and polyethyleneimine.

7. The targeting vector according to claim 5, characterized in that The second molecule is selected from VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof.

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

9. The targeting vector according to claim 8, characterized in that The first molecule further comprises an extracellular hinge region.

10. The targeting vector according to claim 1, wherein The endocytic receptors are selected from the group consisting of 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, Collagen IV, Periostin, CEACAM, c-MET, TDGF1, IGF1R, Mesothelin, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, glycosphingolipid, TfR, Gan glioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, ASGPR.

11. The targeting vector according to claim 1, wherein 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.

12. The targeting vector according to claim 11, characterized in that The viral envelope protein mutates, which weakens its receptor recognition ability.

13. The targeting vector according to claim 12, characterized in that The mutation is a deletion or a substitution.

14. The targeting vector according to claim 12 or 13, characterized in that The viral envelope protein is selected from the group consisting of 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 targeting vector according to claim 14, characterized in that The envelope glycoprotein VSVG of the Indiana strain of vesicular stomatitis virus comprises one or more combinations of the following site mutations: substitution / deletion of H8, substitution / deletion of N9, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of K50, substitution / deletion of A51, substitution / deletion of S183, substitution / deletion of S179, substitution / deletion of N180, substitution / deletion of I182, substitution / deletion of M184, substitution / deletion of Y209, substitution / deletion of Loss, substitution / deletion of I347, substitution / deletion of T350, substitution / deletion of T352, substitution / deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353.

16. The targeting vector according to claim 14, characterized in that Compared with the entire domain of 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 mutations corresponding to the following sites: substitution / deletion of H8, substitution / deletion of N9, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of K50, substitution / deletion of A51, substitution / deletion of S183, substitution / deletion of S179, substitution / deletion of N180, substitution / deletion of I182, substitution / deletion of M184, substitution / deletion of Y209 Loss, substitution / deletion of I347, substitution / deletion of T350, substitution / deletion of T352, substitution / deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353.

17. The targeting vector according to claim 11, characterized in that The transmembrane protein is CD7 antibody, CD19 antibody, CD33 antibody, ASGRP antibody or ligand, Mesothelin antibody, HER2 antibody.

18. The targeting vector according to claim 1, wherein The substance is at least one of small molecule compounds, proteins, polypeptides, RNA, and DNA.

19. The targeting vector according to claim 1, wherein The target cells are lymphocytes, myeloid cells, hematopoietic stem / progenitor cells or non-blood cells.

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

21. A method for preparing the targeting vector according to any one of claims 1 to 20, comprising the following steps: Design the first molecule based on the endocytic receptor of the target cell, Select the second molecule, The first molecule, the second molecule and the substance carried by the carrier are assembled to prepare a targeting carrier.

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 is used to deliver small molecule compounds, proteins, polypeptides, RNA or DNA.

24. A method of introducing a substance into a cell, the method comprising: The cell is contacted with the targeting vector according to any one of claims 1 to 20.

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

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

27. The method according to claim 24, wherein the cells are T cells, NK cells, B cells, macrophages, granulocytes, dendritic cells, hematopoietic stem cells, hepatocytes, pancreatic islet cells, nerve cells, or muscle cells.

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

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

30. A composition according to claim 29 for use as a medicament.

31. The composition according to claim 30, for use in gene therapy, immunotherapy, cell therapy, treatment of gene defect diseases, treatment of autoimmune diseases, treatment of infectious diseases, and treatment of cancer; the cancer includes blood cancer and solid cancer.

32. A method for treating a disease in a subject, characterized in that Administering 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 to a subject.

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