Mutant vesicular stomatitis virus G glycoprotein and targeting vector

Abstract

The present invention provides a mutant vesicular stomatitis virus G glycoprotein (VSVG) and a targeting vector, relating to the area of ​​vector delivery. The present invention provides an envelope glycoprotein of vesicular stomatitis virus having a first mutation and a second mutation, wherein the first mutation prevents the envelope glycoprotein from mediating complement-mediated inactivation, and the second mutation reduces the envelope glycoprotein's ability to be recognized by a receptor. The present invention further provides a targeting vector comprising a first molecule that binds to an endocytosis receptor of a target cell, and a second molecule that promotes the release of a substance carried on the targeting vector into the cytoplasm rather than causing complement inactivation, wherein the second molecule promotes the occurrence of endosomal or lysosomal escape by the targeting vector and prevents the targeting vector from being inactivated by complement.

Description

Technical Field

[0001] The present invention belongs to the field of vector delivery, and specifically relates to a mutant vesicular stomatitis virus G glycoprotein and a targeting vector. Cross-reference to Related Applications This application claims the priority of a Chinese patent application filed on April 10, 2023, with the application number 2023103753276 and the invention title "Mutant Vesicular Stomatitis Virus G Glycoprotein and Targeting Vector", the entire content of which is incorporated herein by reference in its entirety. Citation of a Sequence Listing Submitted in Electronic Form This application includes a sequence listing in XML format submitted in electronic form, which is incorporated herein by reference in its entirety. The sequence listing was created on April 10, 2024 and is named "JYSW-PA-PCT-NO-06-segl.xml".

Background Art

[0002] The complement system is composed of a series of proteins and belongs to a part of the innate immune system. Complement (C) exists on the surface of normal human and animal sera, tissue fluids and cell membranes, has enzymatic activity after activation, and can generate a complex cascade reaction. The complement system is initiated by the mutual cleavage of a series of enzymes, and finally forms a membrane attack complex similar to pores on the surface of target microorganisms, causing the microorganisms to rupture and die. Complement components can be activated by antigen complexes or antibodies, and remove immune complexes through cell lysis, opsonization, phagocytosis and mediated inflammatory reactions, and express corresponding biological functions. Complement is a functional system and an action amplification system that is widely involved in the defense reaction of organisms against microbial infections and immune regulation, and also mediates immune pathological damage reactions and has important biological effects in the body.

[0003] Complement components that guide regulatory action can exist in soluble or membrane-bound forms and mainly include properdin (P factor), C1 inhibitor (C1INH), factor I, factor H, C4 binding protein (C4BP), S protein, SP40 / 40, membrane cofactor protein (MCP), decay accelerating factor (DAF), homologous restriction factor (HRF), and membrane inhibitor of reactive lysis (MIRL). [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Currently, vectors based on vesicular stomatitis virus G glycoprotein (VSVG) are already widely used in clinical treatment, such as in chimeric antigen receptor T-cell therapy (CAR-T), where VSVG-containing late-onset viruses infect T cells while incorporating polynucleotides encoding CAR molecules to produce CAR-T cells. However, VSVG viral vectors are identified and inactivated by complement after entering the serum, making it difficult for them to effectively reach target cells and exert their effects. Therefore, they cannot be directly used for intravenous in vivo treatment. This is a common problem that also exists with other targeting vectors.

[0005] Therefore, there is a need to modify conventional delivery vectors to prevent them from being inactivated by complement and to improve the effectiveness of delivery. [Means for solving the problem]

[0006] A first aspect of the present invention provides an envelope glycoprotein of a vesicular stomatitis virus having a first mutation and a second mutation, wherein the first mutation prevents the envelope glycoprotein from mediating complement-mediated inactivation, and the second mutation reduces the envelope glycoprotein's ability to be recognized by receptors.

[0007] In one specific proposal, the first mutation includes an amino acid sequence in the ectodomain of the envelope glycoprotein that is listed in SEQ ID NO:1, or an amino acid sequence that has at least 50% agreement with the amino acid sequence listed in SEQ ID NO:1, and the amino acid sequence is a) The amino acid located at position 214 of SEQ ID NO:1, b) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 214 of SEQ ID NO:1, c) The amino acid located at position 352 of SEQ ID NO:1, d) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 352 of SEQ ID NO:1, e) The amino acid located at position 50 of SEQ ID NO:1, f) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to the 50th position of SEQ ID NO:1, g) The amino acid located at position 146 of SEQ ID NO:1, h) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 146 of SEQ ID NO:1 It contains a mutation in at least one of the amino acids.

[0008] The ectodomain of the envelope glycoprotein (Indiana-VSVG) of the Indiana virus strain of the genus Becyclovirus contains the amino acid sequence shown in SEQ ID NO:1.

[0009] In one specific proposal, the mutation is an insertion, deletion, or substitution.

[0010] In one specific alternative, the amino acid sequence is: a) The amino acid located at position 214 of SEQ ID NO:1, b) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 214 of SEQ ID NO:1, c) The amino acid located at position 352 of SEQ ID NO:1, d) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 352 of SEQ ID NO:1, e) The amino acid located at position 50 of SEQ ID NO:1, f) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to the 50th position of SEQ ID NO:1, g) The amino acid located at position 146 of SEQ ID NO:1, h) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 146 of SEQ ID NO:1 This includes the substitution of at least one amino acid among them.

[0011] In one specific proposal, the amino acid sequence includes one or more combinations of the following mutations: substitution at T214, substitution at T352, substitution at K50, and substitution at S146.

[0012] In one specific proposal, the amino acid sequence includes one or more combinations of mutations in which the 214th amino acid is replaced from threonine T to asparagine N, and / or the 352nd amino acid is replaced from threonine T to alanine, and / or the 50th amino acid is replaced from lysine K to threonine T, and / or the 146th amino acid is replaced from serine S to threonine T.

[0013] In one specific alternative, the amino acid sequence is: (1) The 214th amino acid is replaced from threonine T to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A. (2) The 214th amino acid is replaced from threonine T to asparagine N, the 352nd amino acid is replaced from threonine T to alanine A, the 50th amino acid is replaced from lysine K to threonine T, and the 146th amino acid is replaced from serine S to threonine T. It contains one of the mutations.

[0014] In one specific proposal, the ectodomain of the Cocal virus envelope glycoprotein has an amino acid sequence that is at least approximately 50% identical to the amino acid sequence SEQ ID NO:1, as shown in SEQ ID NO:10. KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSV AVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAA AKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPN GILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK Includes.

[0015] The ectodomain of the envelope glycoprotein (Cocal-VSVG) of the Cocal virus, i.e., the Cocal virus strain of the genus Vesiculovirus, contains an amino acid sequence as shown in SEQ ID NO:10, and the amino acid sequence as shown in SEQ ID NO:10 has at least 50% identity with the amino acid sequence as shown in SEQ ID NO:1, i.e., the ectodomain of Indiana-VSVG.

[0016] In a specific embodiment, SEQ ID NO:10 a) the amino acid located at position 214 of SEQ ID NO:10, b) the amino acid at the position corresponding to position 214 of SEQ ID NO:10 after performing an optimal global alignment with SEQ ID NO:10, c) the amino acid located at position 352 of SEQ ID NO:10, d) the amino acid at the position corresponding to position 352 of SEQ ID NO:10 after performing an optimal global alignment with SEQ ID NO:10, e) the amino acid located at position 50 of SEQ ID NO:10, f) the amino acid at the position corresponding to position 50 of SEQ ID NO:10 after performing an optimal global alignment with SEQ ID NO:10, g) the amino acid located at position 146 of SEQ ID NO:10, h) the amino acid at the position corresponding to position 146 of SEQ ID NO:10 after performing an optimal global alignment with SEQ ID NO:10 contains a mutation of at least one of the amino acids.

[0017] In a specific embodiment, the mutation is an insertion, deletion or substitution.

[0018] In a specific embodiment, SEQ ID NO:10 a) the amino acid located at position 214 of SEQ ID NO:10, b) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 214 of SEQ ID NO:10, c) The amino acid located at position 352 of SEQ ID NO:10, d) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 352 of SEQ ID NO:10, e) The amino acid located at position 50 of SEQ ID NO:10, f) After performing optimal global alignment with SEQ ID NO:10, the amino acid at the position corresponding to the 50th position of SEQ ID NO:10, g) The amino acid located at position 146 of SEQ ID NO:10, h) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 146 of SEQ ID NO:10 This includes the substitution of at least one amino acid among them.

[0019] One specific proposal suggests that SEQ ID NO:10 contains one or more combinations of the following mutations: substitution of K214, substitution of T352, substitution of K50, and substitution of S146.

[0020] In one specific example, SEQ ID NO:10 contains one or more combinations of mutations in which the 214th amino acid is replaced from lysine K to asparagine N, and / or the 352nd amino acid is replaced from threonine T to alanine, and / or the 50th amino acid is replaced from lysine K to threonine T, and / or the 146th amino acid is replaced from serine S to threonine T.

[0021] The aforementioned SEQ ID NO:10 is, (1) Replacement of K214 and T352, (2) Replacement of K214, T352, K50 and S146 This includes one of the mutations.

[0022] In one specific alternative, SEQ ID NO:10 is, The 214th amino acid is substituted from lysine K to asparagine N, and the 352nd amino acid is substituted from threonine T to alanine A. The 214th amino acid is substituted from lysine K to asparagine N, the 352nd amino acid is substituted from threonine T to alanine A, the 50th amino acid is substituted from lysine K to threonine T, and the 146th amino acid is substituted from serine S to threonine T. It contains one of the mutations.

[0023] In some specific schemes, the envelope glycoprotein of the bullous stomatitis virus having a first mutation and a second mutation is such that, by any of the aforementioned first mutations, the ability of the envelope glycoprotein to antagonize complement-mediated inactivation is enhanced compared to before the occurrence of any of the aforementioned first mutations, and by the second mutations, the ability of the envelope glycoprotein to be recognized by its receptor is reduced compared to before the occurrence of the second mutation.

[0024] In some specific schemes, the aforementioned first mutation enhances the ability of the envelope glycoprotein to resist complement-mediated inactivation compared to before the mutation occurred.

[0025] In some specific designs, the receptor is a receptor for an envelope glycoprotein.

[0026] In some specific designs, the receptor for the envelope glycoprotein is the LDL-R (low-density lipoprotein receptor).

[0027] In one specific proposal, the second mutation includes an amino acid sequence in the ectodomain of the envelope glycoprotein that is listed in SEQ ID NO:1, or an amino acid sequence that has at least 50% agreement with the amino acid sequence listed in SEQ ID NO:1, and the amino acid sequence is 1) Substitution / deletion located at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T3 Substitutions / deletions located at position 52, substitutions / deletions located at E353, substitutions / deletions located at R354, deletions of amino acids located at positions 1-18, deletions of amino acids located at positions 19-36, deletions of amino acids located at positions 37-51, deletions of amino acids located at positions 314-384, deletions of amino acids located at positions 321-374, deletions of amino acids located at positions 331-364, deletions of amino acids located at positions 344-354, deletions of amino acids located at positions 345-353, 2) After performing optimal global alignment with SEQ ID NO:1, replace / delete at the position corresponding to H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, and T350. Substitutions / deletions located at positions T352, E353, R354, deletions at positions 1-18, deletions at positions 19-36, deletions at positions 37-51, deletions at positions 314-384, deletions at positions 321-374, deletions at positions 331-364, deletions at positions 344-354, deletions at positions 345-353 It contains a mutation in at least one of the amino acids.

[0028] In some specific schemes, the amino acid sequence is: 1) Substitutions located at H8, N9, Q10, K47, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, R354, deletions, deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353. 2) After performing optimal global alignment with SEQ ID NO:1, substitutions are made at the positions corresponding to H8, N9, Q10, K47, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, and T352. Substitutions, substitutions at the position corresponding to E353, substitutions at the position corresponding to R354, deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1-18, deletions at the positions corresponding to amino acids 19-36, deletions at the positions corresponding to amino acids 37-51, deletions at the positions corresponding to amino acids 314-384, deletions at the positions corresponding to amino acids 321-374, deletions at the positions corresponding to amino acids 331-364, deletions at the positions corresponding to amino acids 344-354, deletions at the positions corresponding to amino acids 345-353 It contains a mutation in at least one of the amino acids.

[0029] In one specific proposal, the amino acid sequence includes one or more combinations of mutations, namely substitutions / deletions of K47 and substitutions / deletions of R354.

[0030] In one specific proposal, the amino acid sequence further includes one or more combinations of mutations in which the 47th amino acid is replaced from lysine K to glutamine Q, the 354th amino acid is replaced from arginine R to glutamine Q, the 47th amino acid is deleted, and the 354th amino acid is deleted.

[0031] In one specific alternative, the amino acid sequence is: (1) Substitution of K47, (2) Replacement of R354 This includes one of the mutations.

[0032] In one specific alternative, the amino acid sequence is: (1) The 47th amino acid is replaced from lysine K to glutamine Q. (2) The 354th amino acid is replaced from arginine R to glutamine Q. It contains one of the mutations.

[0033] In one specific alternative, the amino acid sequence is: Deletion of amino acids located at positions 331-364 of SEQ ID NO:1, deletion of amino acids located at positions 344-354, substitution at K47, deletion at K47, deletion at R354, substitution at R354, After performing optimal global alignment with SEQ ID NO:1, deletes at positions corresponding to amino acids 331-364 of SEQ ID NO:1, deletes at positions corresponding to amino acids 344-354, substitutions at the position corresponding to K47, deletes at the position corresponding to K47, deletes at the position corresponding to R354, and substitutions at the position corresponding to R354 are performed. It contains at least one of the following amino acid mutations.

[0034] In one specific alternative, the amino acid sequence is: Deletion of amino acids located at positions 331-364 of SEQ ID NO:1, deletion of amino acids located at positions 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion of arginine R at position 354, deletion of lysine at position 47, and After performing optimal global alignment with SEQ ID NO:1, deletions were made at positions corresponding to amino acids 331-364 of SEQ ID NO:1, deletions at positions corresponding to amino acids 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion at the position corresponding to arginine R (amino acid 354), and deletion at the position corresponding to lysine (amino acid 47). It contains at least one of the following amino acid mutations.

[0035] In one specific alternative, the amino acid sequence is: Deletion of lysine, the amino acid located at position 47 of SEQ ID NO:1 After performing optimal global alignment with SEQ ID NO:1, the deletion at the position corresponding to lysine, the 47th amino acid of SEQ ID NO:1 This includes a mutation in the amino acid.

[0036] In one specific proposal, the second mutation is an amino acid sequence, SEQ ID NO:10, which has at least 50% agreement with the aforementioned amino acid sequence SEQ ID NO:1. Substitution or deletion located at Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350 This includes deletions, substitutions or deletions located at T352, substitutions or deletions located at E353, substitutions or deletions located at R354, deletions of amino acids at positions 1-18, deletions of amino acids at positions 19-36, deletions of amino acids at positions 37-51, deletions of amino acids at positions 314-384, deletions of amino acids at positions 321-374, deletions of amino acids at positions 331-364, deletions of amino acids at positions 344-354, and deletions of amino acids at positions 345-353. After performing optimal global alignment with SEQ ID NO:10, substitutions or deletions at the positions corresponding to Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, and S350 are replaced or deleted. Substitutions or deletions at the corresponding positions, substitutions or deletions at the position corresponding to T352, substitutions or deletions at the position corresponding to E353, substitutions or deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1-18, deletions at the positions corresponding to amino acids 19-36, deletions at the positions corresponding to amino acids 37-51, deletions at the positions corresponding to amino acids 314-384, deletions of amino acids at positions 321-374, deletions at the positions corresponding to amino acids 331-364, deletions at the positions corresponding to amino acids 344-354, deletions at the positions corresponding to amino acids 345-353 It contains a mutation in at least one of the amino acids.

[0037] In one specific alternative, SEQ ID NO:10 is, Substitutions located at Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350, T352, E353, R354, deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353. After performing optimal global alignment with SEQ ID NO:10, substitutions are made at the position corresponding to Q8 of SEQ ID NO:10, substitutions are made at the position corresponding to S9, substitutions are made at the position corresponding to Q10, substitutions or deletions are made at the position corresponding to K47, substitutions are made at the position corresponding to K50, substitutions are made at the position corresponding to A51, substitutions are made at the position corresponding to D183, substitutions are made at the position corresponding to A179, substitutions are made at the position corresponding to T180, substitutions are made at the position corresponding to V182, substitutions are made at the position corresponding to T184, substitutions are made at the position corresponding to Y209, substitutions are made at the position corresponding to I347, substitutions are made at the position corresponding to S350, and substitutions are made at the position corresponding to T352. Substitutions at the specified position, substitutions at the position corresponding to E353, substitutions or deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1-18, deletions at the positions corresponding to amino acids 19-36, deletions at the positions corresponding to amino acids 37-51, deletions at the positions corresponding to amino acids 314-384, deletions of amino acids at positions 321-374, deletions at the positions corresponding to amino acids 331-364, deletions at the positions corresponding to amino acids 344-354, deletions at the positions corresponding to amino acids 345-353 It contains a mutation in at least one of the amino acids.

[0038] In one specific alternative, SEQ ID NO:10 is, Deletion of amino acids located at positions 331-364, deletion of amino acids located at positions 344-354, substitution at K47, deletion at K47, deletion at R354, substitution at R354, After performing optimal global alignment with SEQ ID NO:10, deletions at positions corresponding to amino acids 331-364, deletions at positions corresponding to amino acids 344-354, substitutions at the position corresponding to K47, deletions at the position corresponding to K47, deletions at the position corresponding to R354, and substitutions at the position corresponding to R354 were performed. It contains a mutation in at least one of the amino acids.

[0039] In one specific alternative, SEQ ID NO:10 is, Deletion of amino acids located at positions 331-364 of SEQ ID NO:10, deletion of amino acids located at positions 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion of arginine R at position 354, deletion of lysine at position 47, and After performing optimal global alignment with SEQ ID NO:10, the following deletions were made: deletions at positions corresponding to amino acids 331-364 of SEQ ID NO:10, deletions at positions corresponding to amino acids 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion at position corresponding to arginine R at position 354, and deletion at position corresponding to lysine at position 47. It contains a mutation in at least one of the amino acids.

[0040] In one specific alternative, SEQ ID NO:10 is, Deletion of lysine, the amino acid located at position 47 of SEQ ID NO:10. After performing optimal global alignment with SEQ ID NO:10, the deletion at the position corresponding to lysine, the 47th amino acid of SEQ ID NO:10 This includes a mutation in the amino acid.

[0041] In one specific proposal, the ectodomain of the envelope glycoprotein that has produced the first and second mutations contains one of the aforementioned amino acid sequences, such as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0042] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:11, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:11, wherein the amino acid sequence shown in SEQ ID NO:11 is the amino acid sequence after the deletion of lysine, the 47th amino acid of the amino acid sequence shown in SEQ ID NO:1, the substitution of threonine T with asparagine N (compared to before the deletion of K47), and the substitution of threonine T with alanine A (compared to before the deletion of K47).

[0043] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:12, or an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:12, and the amino acid sequence such as that shown in SEQ ID NO:12 is SEQ ID This is the amino acid sequence after the deletion of lysine, the 47th amino acid in the amino acid sequence shown in NO:1, the substitution of threonine T to asparagine N (compared to the sequence before the K47 deletion), the substitution of threonine T to alanine A (compared to the sequence before the K47 deletion), the substitution of lysine K to threonine T (compared to the sequence before the K47 deletion), and the substitution of serine S to threonine T (compared to the sequence before the K47 deletion).

[0044] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:2, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:2, wherein the amino acid sequence shown in SEQ ID NO:2 is the amino acid sequence obtained after the 354th amino acid of the amino acid sequence shown in SEQ ID NO:1 is replaced from arginine R to glutamine Q, the 214th amino acid is replaced from threonine T to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A.

[0045] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:3, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:3, wherein the amino acid sequence shown in SEQ ID NO:3 is the amino acid sequence after the 354th amino acid of the amino acid sequence shown in SEQ ID NO:1 has been replaced from arginine R to glutamine Q, the 214th amino acid has been replaced from threonine T to asparagine N, the 352nd amino acid has been replaced from threonine T to alanine A, the 50th amino acid has been replaced from lysine K to threonine T, and the 146th amino acid has been replaced from serine S to threonine T.

[0046] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:15, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to the amino acid sequence shown in SEQ ID NO:15, wherein the amino acid sequence shown in SEQ ID NO:15 is the amino acid sequence after the deletion of arginine R, which is the 354th amino acid of the amino acid sequence shown in SEQ ID NO:1, the substitution of threonine T with asparagine N (compared to before the deletion of arginine R, the 354th amino acid), and the substitution of threonine T with alanine A (compared to before the deletion of arginine R, the 352nd amino acid).

[0047] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:16, or an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:16, and the amino acid sequence such as that shown in SEQ ID NO:16 is SEQ ID This is the amino acid sequence after the deletion of arginine R, the 354th amino acid in the amino acid sequence shown in NO:1; (compared to before the deletion of arginine R, the 214th amino acid) being replaced from threonine T to asparagine N; (compared to before the deletion of arginine R, the 354th amino acid) being replaced from threonine T to alanine A; (compared to before the deletion of arginine R, the 354th amino acid) being replaced from lysine K to threonine T; and (compared to before the deletion of arginine R, the 354th amino acid) being replaced from serine S to threonine T.

[0048] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:17, or an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:17, and the amino acid sequence such as that shown in SEQ ID NO:17 is SEQ ID This is the amino acid sequence after the deletion of lysine, the 47th amino acid in the amino acid sequence shown in NO:10, the following changes have occurred: (compared to before the deletion of K47) the 214th amino acid is replaced from lysine K to asparagine N, (compared to before the deletion of K47) the 352nd amino acid is replaced from threonine T to alanine A, (compared to before the deletion of K47) the 50th amino acid is replaced from lysine K to threonine T, and (compared to before the deletion of K47) the 146th amino acid is replaced from serine S to threonine T.

[0049] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:18, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:18, wherein the amino acid sequence shown in SEQ ID NO:18 is the amino acid sequence after the deletion of lysine, the 47th amino acid of the amino acid sequence shown in SEQ ID NO:10, the substitution of lysine K to asparagine N at the 214th amino acid (compared to before the deletion of K47), and the substitution of threonine T to alanine A at the 352nd amino acid (compared to before the deletion of K47).

[0050] In one specific proposal, the ectodomain of the envelope glycoprotein includes an amino acid sequence such as that shown in SEQ ID NO:19, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:19, wherein the amino acid sequence shown in SEQ ID NO:19 is the amino acid sequence obtained after the amino acid sequence shown in SEQ ID NO:10 has been modified in which the 354th amino acid is replaced from arginine R to glutamine Q, the 214th amino acid is replaced from lysine K to asparagine N, the 352nd amino acid is replaced from threonine T to alanine A, the 50th amino acid is replaced from lysine K to threonine T, and the 146th amino acid is replaced from serine S to threonine T.

[0051] In one specific proposal, the ectodomain of the envelope glycoprotein includes an amino acid sequence such as that shown in SEQ ID NO:20, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:20, wherein the amino acid sequence shown in SEQ ID NO:20 is the amino acid sequence obtained by substituting the 354th amino acid of the amino acid sequence shown in SEQ ID NO:10 from arginine R to glutamine Q, substituting the 214th amino acid from lysine K to asparagine N, and substituting the 352nd amino acid from threonine T to alanine A.

[0052] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:13, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:13, wherein the amino acid sequence shown in SEQ ID NO:13 is the amino acid sequence after the deletion of arginine R, the 354th amino acid of the amino acid sequence shown in SEQ ID NO:10, the substitution of lysine K to asparagine N (compared to before the deletion of arginine R, the 354th amino acid), and the substitution of threonine T to alanine A (compared to before the deletion of arginine R, the 352nd amino acid).

[0053] In one specific proposal, the ectodomain of the envelope glycoprotein contains an amino acid sequence such as that shown in SEQ ID NO:14, or an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:14, and the amino acid sequence such as that shown in SEQ ID NO:14 is SEQ ID This is the amino acid sequence after the deletion of arginine R, the 354th amino acid in the amino acid sequence shown in NO:10; (compared to before the deletion of arginine R, the 354th amino acid) the 214th amino acid is replaced from lysine K to asparagine N; (compared to before the deletion of arginine R, the 354th amino acid) the 352nd amino acid is replaced from threonine T to alanine A; (compared to before the deletion of arginine R, the 354th amino acid) the 50th amino acid is replaced from lysine K to threonine T; and (compared to before the deletion of arginine R, the 354th amino acid) the 146th amino acid is replaced from serine S to threonine T.

[0054] Another aspect of the present invention provides a nucleic acid molecule encoding an envelope glycoprotein as described in the present invention.

[0055] Another aspect of the present invention provides a vector comprising a nucleic acid molecule as described in the present invention or expressing an envelope glycoprotein as described in the present invention.

[0056] In one specific proposal, the vector is a slow-acting viral vector or a retroviral vector.

[0057] Another aspect of the present invention provides a targeting vector comprising a first molecule that binds to an endocytosis receptor of a target cell, and a second molecule that facilitates the release of a substance carried on the targeting vector into the cytoplasm, without deactivating complement.

[0058] The action of the second molecule is to promote the endosomal or lysosomal escape of the targeting vector and to prevent the targeting vector from being inactivated by complement, which is a mutant of the envelope glycoprotein described in the present invention, in particular a mutant of the envelope glycoprotein of vesicular stomatitis virus, the mutant comprising a first mutation that prevents the envelope glycoprotein from being inactivated by complement.

[0059] The first mutation includes an amino acid sequence in the ectodomain of the envelope glycoprotein that is listed in SEQ ID NO:1, or an amino acid sequence that has at least 50% agreement with the amino acid sequence listed in SEQ ID NO:1, wherein the amino acid sequence is a) The amino acid located at position 214 of SEQ ID NO:1, b) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 214 of SEQ ID NO:1, c) The amino acid located at position 352 of SEQ ID NO:1, d) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 352 of SEQ ID NO:1, e) The amino acid located at position 50 of SEQ ID NO:1, f) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to the 50th position of SEQ ID NO:1, g) The amino acid located at position 146 of SEQ ID NO:1, h) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 146 of SEQ ID NO:1 It contains a mutation in at least one of the amino acids.

[0060] In one specific proposal, the mutation is an insertion, deletion, or substitution.

[0061] In one specific alternative, the amino acid sequence is: a) The amino acid located at position 214 of SEQ ID NO:1, b) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 214 of SEQ ID NO:1, c) The amino acid located at position 352 of SEQ ID NO:1, d) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 352 of SEQ ID NO:1, e) The amino acid located at position 50 of SEQ ID NO:1, f) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to the 50th position of SEQ ID NO:1, g) The amino acid located at position 146 of SEQ ID NO:1, h) After performing optimal global alignment with SEQ ID NO:1, the amino acid at position 146 of SEQ ID NO:1 This includes the substitution of at least one amino acid among them.

[0062] In one specific proposal, the amino acid sequence includes one or more combinations of the following mutations: substitution at T214, substitution at T352, substitution at K50, and substitution at S146.

[0063] In one specific proposal, the amino acid sequence includes one or more combinations of mutations in which the 214th amino acid is replaced from threonine T to asparagine N, and / or the 352nd amino acid is replaced from threonine T to alanine, and / or the 50th amino acid is replaced from lysine K to threonine T, and / or the 146th amino acid is replaced from serine S to threonine T.

[0064] In one specific alternative, the amino acid sequence is: (1) The 214th amino acid is replaced from threonine T to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A. (2) The 214th amino acid is replaced from threonine T to asparagine N, the 352nd amino acid is replaced from threonine T to alanine A, the 50th amino acid is replaced from lysine K to threonine T, and the 146th amino acid is replaced from serine S to threonine T. It contains one of the mutations.

[0065] In one specific proposal, the ectodomain of the Cocal virus envelope glycoprotein has an amino acid sequence that is at least approximately 50% identical to the amino acid sequence SEQ ID NO:1, as shown in SEQ ID NO:10. KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSV AVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAA AKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPN GILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK Includes.

[0066] In one specific proposal, the first mutation described above has SEQ ID NO:10, a) The amino acid located at position 214 of SEQ ID NO:10, b) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 214 of SEQ ID NO:10, c) The amino acid located at position 352 of SEQ ID NO:10, d) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 352 of SEQ ID NO:10, e) The amino acid located at position 50 of SEQ ID NO:10, f) After performing optimal global alignment with SEQ ID NO:10, the amino acid at the position corresponding to the 50th position of SEQ ID NO:10, g) The amino acid located at position 146 of SEQ ID NO:10, h) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 146 of SEQ ID NO:10 This includes having a mutation in at least one of the amino acids.

[0067] In one specific proposal, the mutation is an insertion, deletion, or substitution.

[0068] In one specific alternative, SEQ ID NO:10 is, a) The amino acid located at position 214 of SEQ ID NO:10, b) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 214 of SEQ ID NO:10, c) The amino acid located at position 352 of SEQ ID NO:10, d) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 352 of SEQ ID NO:10, e) The amino acid located at position 50 of SEQ ID NO:10, f) After performing optimal global alignment with SEQ ID NO:10, the amino acid at the position corresponding to the 50th position of SEQ ID NO:10, g) The amino acid located at position 146 of SEQ ID NO:10, h) After performing optimal global alignment with SEQ ID NO:10, the amino acid at position 146 of SEQ ID NO:10 This includes the substitution of at least one amino acid among them.

[0069] One specific proposal is that SEQ ID NO:10 contains one or more combinations of mutations from among the substitutions of K214, T352, K50, and S146.

[0070] In one specific example, SEQ ID NO:10 contains one or more combinations of mutations in which the 214th amino acid is replaced from lysine K to asparagine N, and / or the 352nd amino acid is replaced from threonine T to alanine, and / or the 50th amino acid is replaced from lysine K to threonine T, and / or the 146th amino acid is replaced from serine S to threonine T.

[0071] The aforementioned SEQ ID NO:10 is, (1) Replacement of K214 and T352, (2) Replacement of K214, T352, K50 and S146 This includes one of the mutations.

[0072] In one specific alternative, SEQ ID NO:10 is, The 214th amino acid is substituted from lysine K to asparagine N, and the 352nd amino acid is substituted from threonine T to alanine A. The 214th amino acid is substituted from lysine K to asparagine N, the 352nd amino acid is substituted from threonine T to alanine A, the 50th amino acid is substituted from lysine K to threonine T, and the 146th amino acid is substituted from serine S to threonine T. It contains one of the mutations.

[0073] In one specific proposal, the action of the second molecule is to promote the endosomal or lysosomal escape of the targeting vector and to enhance the targeting vector's ability to resist complement-mediated inactivation, which is a mutant of the envelope glycoprotein described in the present invention, particularly a mutant of the envelope glycoprotein of vesicular stomatitis virus, the mutant comprising the aforementioned arbitrary first mutation which enhances the ability of the envelope glycoprotein to resist complement-mediated inactivation, and the enhancement of the ability to resist complement-mediated inactivation is compared to before the occurrence of the aforementioned arbitrary first mutation.

[0074] The mutant further includes a second mutation that reduces the discriminative ability of the envelope glycoprotein receptor by weakening or eliminating its ability to bind to LDL-R, thereby allowing the mutant to retain the ability to escape from endosomes or lysosomes, and thus becoming entirely dependent on the first molecule to exert its targeting effect. By possessing the mutated envelope glycoprotein, the targeting ability of the vector is further increased, making it possible for the vector to infect only specific target cells. In one specific proposal, the second molecule is a VSVG that has undergone an insertion, deletion, or substitution mutation, resulting in a loss of the ability of VSVG to bind to LDL-R, but without simultaneously affecting the lysosome escape ability of VSVG.

[0075] The second mutation includes an amino acid sequence in the ectodomain of the envelope glycoprotein that is listed in SEQ ID NO:1, or an amino acid sequence that has at least 50% agreement with the amino acid sequence listed in SEQ ID NO:1, wherein the amino acid sequence is 1) Substitution / deletion located at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T3 Substitutions / deletions located at position 52, substitutions / deletions located at E353, substitutions / deletions located at R354, deletions of amino acids located at positions 1-18, deletions of amino acids located at positions 19-36, deletions of amino acids located at positions 37-51, deletions of amino acids located at positions 314-384, deletions of amino acids located at positions 321-374, deletions of amino acids located at positions 331-364, deletions of amino acids located at positions 344-354, deletions of amino acids located at positions 345-353, 2) After performing optimal global alignment with SEQ ID NO:1, replace / delete at the position corresponding to H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, and T350. Substitutions / deletions located at positions T352, E353, R354, deletions at positions 1-18, deletions at positions 19-36, deletions at positions 37-51, deletions at positions 314-384, deletions at positions 321-374, deletions at positions 331-364, deletions at positions 344-354, deletions at positions 345-353 It contains a mutation in at least one of the amino acids.

[0076] In some specific schemes, the amino acid sequence is: 1) Substitutions located at H8, N9, Q10, K47, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, R354, deletions, deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353. 2) After performing optimal global alignment with SEQ ID NO:1, substitutions are made at the positions corresponding to H8, N9, Q10, K47, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, and T352. Substitutions, substitutions at the position corresponding to E353, substitutions at the position corresponding to R354, deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1-18, deletions at the positions corresponding to amino acids 19-36, deletions at the positions corresponding to amino acids 37-51, deletions at the positions corresponding to amino acids 314-384, deletions at the positions corresponding to amino acids 321-374, deletions at the positions corresponding to amino acids 331-364, deletions at the positions corresponding to amino acids 344-354, deletions at the positions corresponding to amino acids 345-353 It contains a mutation in at least one of the amino acids.

[0077] In one specific proposal, the amino acid sequence further includes one or more mutations, namely substitutions / deletions of K47 and substitutions / deletions of R354.

[0078] In one specific proposal, the amino acid sequence further includes one or more combinations of mutations in which the 47th amino acid is replaced from lysine K to glutamine Q, the 354th amino acid is replaced from arginine R to glutamine Q, the 47th amino acid is deleted, and the 354th amino acid is deleted.

[0079] In one specific alternative, the amino acid sequence is: (1) Substitution of K47, (2) Replacement of R354 This includes one of the mutations.

[0080] In one specific alternative, the amino acid sequence is: (1) The 47th amino acid is replaced from lysine K to glutamine Q. (2) The 354th amino acid is replaced from arginine R to glutamine Q. It contains one of the mutations.

[0081] In one specific alternative, the amino acid sequence is: Deletion of amino acids located at positions 331-364 of SEQ ID NO:1, deletion of amino acids located at positions 344-354, substitution at K47, deletion at K47, deletion at R354, substitution at R354, After performing optimal global alignment with SEQ ID NO:1, deletes at positions corresponding to amino acids 331-364 of SEQ ID NO:1, deletes at positions corresponding to amino acids 344-354, substitutions at the position corresponding to K47, deletes at the position corresponding to K47, deletes at the position corresponding to R354, and substitutions at the position corresponding to R354 are performed. It contains at least one of the following amino acid mutations.

[0082] In one specific alternative, the amino acid sequence is: Deletion of amino acids located at positions 331-364 of SEQ ID NO:1, deletion of amino acids located at positions 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion of arginine R at position 354, deletion of lysine at position 47, and After performing optimal global alignment with SEQ ID NO:1, deletions were made at positions corresponding to amino acids 331-364 of SEQ ID NO:1, deletions at positions corresponding to amino acids 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion at the position corresponding to arginine R (amino acid 354), and deletion at the position corresponding to lysine (amino acid 47). It contains at least one of the following amino acid mutations.

[0083] In one specific proposal, the aforementioned amino acid sequence (1) has a deletion of lysine, which is the 47th amino acid in SEQ ID NO:1. After performing optimal global alignment with SEQ ID NO:1, the deletion at the position corresponding to lysine, the 47th amino acid of SEQ ID NO:1 This includes a mutation in the amino acid.

[0084] In one specific proposal, the second mutation is an amino acid sequence, SEQ ID NO:10, which has at least a 50% agreement with the aforementioned amino acid sequence SEQ ID NO:1. Substitution or deletion located at Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350 This includes deletions, substitutions or deletions located at T352, substitutions or deletions located at E353, substitutions or deletions located at R354, deletions of amino acids at positions 1-18, deletions of amino acids at positions 19-36, deletions of amino acids at positions 37-51, deletions of amino acids at positions 314-384, deletions of amino acids at positions 321-374, deletions of amino acids at positions 331-364, deletions of amino acids at positions 344-354, and deletions of amino acids at positions 345-353. After performing optimal global alignment with SEQ ID NO:10, substitutions or deletions at the positions corresponding to Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, and S350 are replaced or deleted. Substitutions or deletions at the corresponding positions, substitutions or deletions at the position corresponding to T352, substitutions or deletions at the position corresponding to E353, substitutions or deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1-18, deletions at the positions corresponding to amino acids 19-36, deletions at the positions corresponding to amino acids 37-51, deletions at the positions corresponding to amino acids 314-384, deletions of amino acids at positions 321-374, deletions at the positions corresponding to amino acids 331-364, deletions at the positions corresponding to amino acids 344-354, deletions at the positions corresponding to amino acids 345-353 This includes having a mutation in at least one of the amino acids.

[0085] In one specific alternative, SEQ ID NO:10 is, Substitutions located at Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350, T352, E353, R354, deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353. After performing optimal global alignment with SEQ ID NO:10, substitutions are made at the position corresponding to Q8 of SEQ ID NO:10, substitutions are made at the position corresponding to S9, substitutions are made at the position corresponding to Q10, substitutions or deletions are made at the position corresponding to K47, substitutions are made at the position corresponding to K50, substitutions are made at the position corresponding to A51, substitutions are made at the position corresponding to D183, substitutions are made at the position corresponding to A179, substitutions are made at the position corresponding to T180, substitutions are made at the position corresponding to V182, substitutions are made at the position corresponding to T184, substitutions are made at the position corresponding to Y209, substitutions are made at the position corresponding to I347, substitutions are made at the position corresponding to S350, and substitutions are made at the position corresponding to T352. Substitutions at the specified position, substitutions at the position corresponding to E353, substitutions or deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1-18, deletions at the positions corresponding to amino acids 19-36, deletions at the positions corresponding to amino acids 37-51, deletions at the positions corresponding to amino acids 314-384, deletions of amino acids at positions 321-374, deletions at the positions corresponding to amino acids 331-364, deletions at the positions corresponding to amino acids 344-354, deletions at the positions corresponding to amino acids 345-353 It contains a mutation in at least one of the amino acids.

[0086] In one specific alternative, SEQ ID NO:10 is, Deletion of amino acids located at positions 331-364, deletion of amino acids located at positions 344-354, substitution at K47, deletion at K47, deletion at R354, substitution at R354, After performing optimal global alignment with SEQ ID NO:10, deletions at positions corresponding to amino acids 331-364, deletions at positions corresponding to amino acids 344-354, substitutions at the position corresponding to K47, deletions at the position corresponding to K47, deletions at the position corresponding to R354, and substitutions at the position corresponding to R354 were performed. It contains a mutation in at least one of the amino acids.

[0087] In one specific alternative, SEQ ID NO:10 is, Deletion of amino acids located at positions 331-364 of SEQ ID NO:10, deletion of amino acids located at positions 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion of arginine R at position 354, deletion of lysine at position 47, and After performing optimal global alignment with SEQ ID NO:10, the following deletions were made: deletions at positions corresponding to amino acids 331-364 of SEQ ID NO:10, deletions at positions corresponding to amino acids 344-354, substitution of lysine K to glutamine Q at position 47, substitution of arginine R to glutamine Q at position 354, deletion at position corresponding to arginine R at position 354, and deletion at position corresponding to lysine at position 47. It contains a mutation in at least one of the amino acids.

[0088] In one specific alternative, SEQ ID NO:10 is, Deletion of lysine, the amino acid located at position 47 of SEQ ID NO:10. After performing optimal global alignment with SEQ ID NO:10, the deletion at the position corresponding to lysine, the 47th amino acid of SEQ ID NO:10 This includes a mutation in the amino acid.

[0089] In one specific proposal, the ectodomain of the envelope glycoprotein (second molecule) that has produced the first and second mutations contains one of the aforementioned amino acid sequences, such as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0090] In one specific proposal, the reduction in the ability to identify the aforementioned receptor is compared to the state before any second mutation occurred.

[0091] The targeting vector is an enveloped virus, such as a retroviral vector or a slow-acting viral vector, preferably a VSV vector, the second molecule is the viral envelope glycoprotein, preferably the vesicular stomatitis virus envelope glycoprotein VSVG and its variants, and the first molecule is not part of the viral envelope glycoprotein.

[0092] Viral envelope glycoproteins like VSVG can facilitate membrane fusion between the viral envelope and endosomes / lysosomes within endosomes / lysosomes, thereby promoting the release of substances carried by the vector.

[0093] A mutant is a mutant that has at least 75% similarity to the amino acid sequence of a non-mutant (wild-type) mutant. "At least 75% similarity" means that the amino acid sequence is 70%, 71%, 72%, 73%, 74%, 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% homology to the amino acid sequence of a non-mutant (wild-type) mutant.

[0094] The VSVG receptor LDL-R is widely expressed in various cell types, including activated T cells, stem cells, cardiomyocytes, endothelial cells, and other tumor cells. Therefore, pseudotyped late-onset viruses carrying VSVG and its variants can infect various cell types. Although VSVG-based pseudotyped viruses possess the ability to infect a wide range of cells, many cells, such as NK cells and quiescent T cells, do not express or express only low levels of the VSVG receptor LDL-R. In these cases, infection is difficult for VSVG-based pseudotyped viruses. Furthermore, because LDL-R is widely expressed in various cell types, the targeting ability of VSVG-based pseudotyped viruses is insufficient, as many cell types express LDL-R, making it difficult for late-onset viruses to accurately infect only one type.

[0095] The action of the second molecule involves promoting endosomal escape or lysosomal escape of the targeting vector after endocytosis by the target cell, thereby enabling it to enter the cytoplasm. If the targeting vector cannot undergo endosomal or lysosomal escape after endocytosis, it may ultimately be degraded by lysosomes. Therefore, for the substance carried by the vector to be effectively transported into the cytoplasm, the vector must possess the ability to undergo endosomal or lysosomal escape.

[0096] This approach utilizes the endosomal / lysosome escape ability of slow-release viruses and modifies them accordingly. The targeting vector obtained by conjugating the first molecule to the slow-release virus can be actively applied to various situations in various cells. By designing the first molecule based on the cells that need to be targeted, the range of application can be greatly expanded, and the accuracy of targeting can also be improved.

[0097] Endocytosis receptors refer to membrane proteins, such as CD7, CD5, HER2, and mesoserine, that are expressed on the cell surface and can induce endocytosis (also called cell cytosis) after binding to antibodies, ligands, or specific substances. Typically, endocytosis receptors contain domains such as YXXPhi, [D / E]XXXL[L / I], and FXNPXY. However, some membrane proteins, such as the CD8 molecule, do not possess the endocytosis receptor domain and therefore do not have the ability to induce endocytosis. Consequently, when screening for endocytosis domains, the CD8 molecule, whose posterior end of the intracellular sequence interlocks with a random sequence, is often used for screening.

[0098] Since different cells express different specific endocytosis receptors, the first molecule can be designed based on the endocytosis receptor of the cell that needs to be targeted. The vector is then endocytotic by the target cell using an antibody or ligand (part of the first molecule) that specifically binds to the endocytosis receptor, thereby making the vector infectable to certain cells but not to other cells.

[0099] The first molecule, after binding to the endocytosis receptor, induces receptor-mediated endocytosis. Receptor-mediated endocytosis is the process by which cells specifically take in extracellular proteins or other compounds by relying on receptors on their cell surface. Cell surface receptors are highly specific and bind to their corresponding ligands to form complexes. Subsequently, the cell membrane in this region invaginates to form a covered small cavity, which then detaches from the cell membrane to form a covered vesicle, taking in extracellular material into the cell. After entering the cell, the covered vesicle sheds its outer covering and binds to vesicles in the cell's endosomes to form a larger endosome or endosome.

[0100] In one specific proposal, the first molecule comprises a transmembrane peptide portion and an antibody or ligand that binds to the endocytosis receptor of the target cell. In some proposals, the first molecule further comprises an extracellular hinge site. Membrane-expressed proteins generally require a hinge site that facilitates the extension of the membrane protein, which is typically the CD8 hinge site.

[0101] The amino acid sequence of the first molecule, which is not limited as long as it does not affect performance, such as when the first molecule contains a CD33 antibody, can be a protein that is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to the amino acid sequence of the CD33 antibody.

[0102] Endocytosis receptors for different target cells 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, and CD30. These are the usual, somewhat different endocytosis receptors, such as TROP2, FRα, STEAP1, ENPP3, GCC, SLC44A4, NaPi2b, CA9, SC-16, CD142, P-cadherin, PSMA, ED-B, endothelin receptor ETB, TN-C, collagen IV, periostin, CEACAM, c-MET, TDGF1, IGF1R, mesoserine, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, sphingoglycolipid, TfR, ganglioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, and ASGPR.

[0103] In one specific proposal, the endocytosis receptor is CD7.

[0104] In one specific proposal, the antibody is an anti-CD7 single-domain antibody, the HCDR1 domain of the anti-CD7 single-domain antibody contains an amino acid sequence as shown in SEQ ID NO:22, or having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:22, the HCDR2 domain of the anti-CD7 single-domain antibody contains an amino acid sequence as shown in SEQ ID NO:23, or having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:23, and the HCDR3 domain of the anti-CD7 single-domain antibody contains an amino acid sequence as shown in SEQ ID NO:24, or SEQ ID It includes an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in NO:24.

[0105] In one specific proposal, the anti-CD7 single-domain antibody (VHH) includes an amino acid sequence such as that shown in SEQ ID NO:21, or a sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO:21.

[0106] In one specific proposal, the targeting vector is a slow-release viral vector, the first molecule expressed by the slow-release viral vector is a transmembrane protein, and the second molecule expressed by the slow-release viral vector is a viral envelope glycoprotein, the viral envelope glycoprotein having the ability to promote endosomal or lysosomal escape. When the target cell does not express the receptor for the viral envelope glycoprotein, the virus can infect the target cell through endocytosis mediated by the first molecule.

[0107] In one specific proposal, the transmembrane protein is selected from CD7 antibody, CD79B antibody, CD33 antibody, ASGRP antibody or its counterpart, mesoserine antibody, HER2 antibody, and the transmembrane protein may further be a protein homologous to at least one amino acid sequence of the CD8 signal peptide of the CD7 antibody, the heavy chain of TH-69 (VH, SEQ ID NO: 5), GS linkage peptide (linker), the light chain of TH-69 (VL, SEQ ID NO: 7), the hinge region of CD8, or the transmembrane region of CD8 by at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, and the transmembrane protein may further be the CD8 signal peptide of the CD33 antibody, the light chain of gemtuzumab (VL), The protein may be at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to at least one amino acid sequence of the GS linked peptide (linker), gemtuzumab heavy chain (VH), CD8 hinge region, or CD8 transmembrane region, and the transmembrane protein may further be the CD79B antibody CD The protein may be at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to at least one amino acid sequence among the signal peptide, the heavy chain (VH) of SN-8, the GS linking peptide (linker), the light chain (VL) of SN-8, the hinge region of CD8, or the transmembrane region of CD8, and the membrane The transmembrane protein further comprises at least one amino acid sequence from the ASGRP antibody's CD8 signal peptide, ASGRP light chain (VL), GS linker peptide, ASGRP heavy chain (VH), CD8 hinge region, and CD8 transmembrane region, with at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99% of the amino acid sequence.The protein may be 5% homologous, and the transmembrane protein is further approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% homologous to at least one amino acid sequence from among the HER2 antibody's CD8 signal peptide, pertuzumab's light chain (VL), GS linkage peptide (linker), pertuzumab's heavy chain (VH), CD8 hinge region, and CD8 transmembrane region. The transmembrane protein may be a protein that is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to at least one amino acid sequence from among the CD8 signal peptide, PE38 heavy chain (VH), GS linker peptide (linker), PE38 light chain (VL), CD8 hinge region, or CD8 transmembrane region of the mesoserine antibody.

[0108] Taking T cells infected with a slow-onset virus as an example, mutated VSVG and CD7 antibodies expressed on the envelope of the slow-onset virus can mediate endocytosis of the slow-onset virus after the CD7 antibody binds to the CD7 antigen on the surface of the T cell. Subsequently, the mutated VSVG mediates lysosomal escape, thereby achieving targeted infection of T cells by the slow-onset virus. Furthermore, the slow-onset virus infects only T cells that express CD7 and has no ability to infect cells that do not express CD7. In addition, mutated VSVG cannot cause the slow-onset virus to be inactivated by serum complement.

[0109] The substances incorporated into the targeting vector are not limited and can include small molecule compounds such as chimeric antigen receptors (CARs) and TCRs, proteins, polypeptides, RNA, DNA, etc., based on actual needs. In one specific implementation, the substance includes a polynucleotide encoding a chimeric antigen receptor. The type of CAR is not limited and includes an antigen-binding domain. In some implementations, the antigen-binding domain is a single-chain variable region fragment (scFV) including variable heavy and light chain regions, which specifically bind to the appropriate antigen. The scFV is selected from monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, human antibodies, nanoantibodies, and synthetic antibodies. In some implementations, the CAR further includes a transmembrane domain (e.g., a CD8 transmembrane domain) and one or more signaling domains based on an immunoreceptor tyrosine activation motif (ITAM) (e.g., CD3ζ). In some implementations, the CAR includes one or more co-stimulatory domains. The type of co-stimulatory domain of the CAR is not limited. In effect, any known co-stimulatory domain in this region can be used, including but not limited to CD28, 4-1BB, DAP10, and DAP12. In some implementations, the CAR may be a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR. In some implementations, the CAR may be single-specific, double-specific, or multiple-specific.

[0110] Small molecule compounds refer to organic compound molecules with a molecular weight of less than 900 daltons, and are particularly small molecules that can be used as drugs.

[0111] In one specific proposal, the target cells are lymphocytes, myeloid cells, hematopoietic stem cells / hematopoietic cells, or non-blood cells, and in particular, myeloid cells, hematopoietic stem cells / hematopoietic cells, or non-blood cells, including normal cells and tumor cells.

[0112] In another aspect of the present invention, a method for preparing a targeting vector is: The method includes the steps of designing a first molecule based on the endocytosis receptor of the target cell, designing a second molecule, and preparing a targeting vector by loading the first molecule, the second molecule, and the substance to be carried by the vector.

[0113] Designing the first and second molecules involves mutating their amino acid sequences. Designing the first molecule, i.e., modifying the viral surface with an antibody / ligand that can target and identify a specific receptor, restricts its design and receptor binding. However, the envelope glycoprotein of a mutant virus possessing endosomal / lysosome escape ability and complement inactivation ability can, as the second molecule, rely on the first molecule regardless of whether the target cell expresses the receptor for the viral envelope glycoprotein. This allows the virus to target and infect target cells regardless, and since the virus cannot infect other cells, the accuracy of targeting is greatly enhanced, and it is not inactivated by serum complement.

[0114] In some specific schemes, the mutation includes any first mutation and any second mutation as described above.

[0115] To construct an enveloped vector, using VSVG as an example, a plasmid expressing an antibody / ligand that conjugates to an endocytosis receptor is mixed with a slow-release viral packaging plasmid such as psPAX2 and pMD.2G (VSVG or a VSVG mutant) and a vector expressed by a slow-release virus to create a vector that expresses targeting.

[0116] Enveloped vectors, such as the pseudotype slow-release viral vector VSVG, enter cells via clathrin-mediated endocytosis after VSVG binds to LDL-R on the cell membrane surface. Following oxidation in the endosomes formed by endocytosis, the structure of VSVG changes, causing the viral envelope to fuse with the endosomal membrane. This allows the virus to detach from the endosome / lysosome and enter the cell nucleus through the nuclear pore.

[0117] Another aspect of the present invention allows the targeting vector to be used for the delivery of drugs or vaccines, and in particular for the delivery of small molecule compounds, proteins, polypeptides, RNA, or DNA.

[0118] Another aspect of the present invention is a method for introducing a substance into cells, comprising bringing the cells into contact with a targeting vector.

[0119] In one specific proposal, the cells in question are mammalian cells.

[0120] In one specific proposal, the cells are either normal cells or cancer cells.

[0121] In one specific proposal, the cells mentioned are T cells, NK cells, B cells, macrophages, granulocytes, dendritic cells, hematopoietic stem cells, hepatocytes, pancreatic islet cells, nerve cells, and muscle cells.

[0122] In one specific proposal, for example, the targeting vector enters the body and comes into contact with target cells, or the target virus directly infects target cells outside the body, by administration performed inside or outside the body, which may be intravenous, intraperitoneal, intratumor, intraosseous, or intranodule.

[0123] The term "mammal" refers to any species of mammal, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, and domesticated animals.

[0124] Another aspect of the present invention is a host cell comprising or expressing the envelope glycoprotein of the present invention, comprising the nucleic acid molecule of the present invention, or comprising the targeting vector of the present invention.

[0125] Another aspect of the present invention is a composition comprising the envelope glycoprotein, nucleic acid molecule, targeting vector, host cell, or any combination thereof, which can be used as a drug and can be used to prepare drugs for gene therapy, immunotherapy, cell therapy, treatment of genetic diseases, and treatment of cancer.

[0126] Another aspect of the present invention is a method for treating a patient or killing diseased cells in a patient, comprising administering the patient a composition of the present invention.

[0127] In one specific proposal, the disease includes cancer, and the cancer includes blood cancer and solid tumors.

[0128] In one specific proposal, the administration is selected from at least one of the following methods: oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intrafocal, continuous release system, and implantable device.

[0129] The term "patient" refers to a subject who is suffering from a disease, illness or condition, or who is at risk of disease, illness or condition progressing, or who otherwise requires the composition or treatment method of the present invention, and "patient" includes, but is not limited to, humans or non-human mammals such as livestock, agricultural animals or wild animals, as well as birds and aquatic animals.

[0130] Throughout this specification, the terms “several specific solutions,” “one specific solution,” “implementation solution,” “a particular implementation solution,” “a related implementation solution,” “a certain implementation solution,” “another implementation solution,” or “other implementation solutions,” or any combination thereof, refer to a combination of implementation solutions, including the specific features, structures, or characteristics described, which are incorporated into at least one implementation solution of the present invention. Therefore, not all of the aforementioned phrases appearing throughout this specification must specify a corresponding implementation solution. Furthermore, specific features, structures, or characteristics can be combined in one or more implementation solutions in any suitable manner. [Effects of the Invention]

[0131] This approach involves inducing rational mutations in the viral envelope glycoprotein and constructing the envelope glycoprotein as a targeting vector. 1. Effectively enhance the targeting vector's ability to antagonize complement-mediated inactivation, thereby increasing the efficiency of infection in serum. 2. Promote highly efficient targeted delivery within the body. It has the beneficial effect of... [Brief explanation of the drawing]

[0132] [Figure 1] Figure 1 is a schematic diagram of a targeting vector whose envelope contains an anti-CD7 antibody. [Figure 2] Figure 2 is a graph showing the measurement results of the efficiency with which the delayed-onset viruses of VSVG mutants 1, 3, and 4, described in Example 1, infect CD7+ Jurkat cells under conditions where serum is present. [Figure 3] Figure 3 is a graph showing the measurement results of the efficiency with which the delayed-onset viruses of VSVG mutants 5 and 6, described in Example 2, infect CD7+ Jurkat cells under conditions where serum is present. [Modes for carrying out the invention] [Examples]

[0133] A slow-acting virus that targets CD+7 cells was created using a VSVG mutant. In this case, the VSVG mutant possesses a mutation that causes it to lose the ability to recognize receptors and to antagonize the complement inactivation ability. As a result, the vector can efficiently target and infect target cells in the presence of serum.

[0134] 1. Design of anti-CD7 single-domain antibodies expressed on membranes. A membrane-bound anti-CD7 single-domain antibody contains, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-CD7 single-domain antibody (VHH), a CD8 hinge region, and a CD8 transmembrane region. The amino acid sequence of the CD8 signal peptide is SEQ ID NO:4: MALPVTALLLPLALLLHAARP That is the case.

[0135] The HCDR1 region of the aforementioned anti-CD7 single-domain antibody is SEQ ID NO:22 SEQ ID NO:22:GRAFSVYAMA It includes an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in or SEQ ID NO:22.

[0136] The HCDR2 region of the aforementioned anti-CD7 single-domain antibody is SEQ ID NO:23 SEQ ID NO:23:SIAGSSSTYYTN It includes an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in or in SEQ ID NO:23.

[0137] The HCDR3 region of the aforementioned anti-CD7 single-domain antibody is SEQ ID NO:24 SEQ ID NO:24:SPRSNNGRETRHYDY It includes an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in or in SEQ ID NO:24.

[0138] The aforementioned anti-CD7 single-domain antibody (VHH) is SEQ ID NO:21 SEQ ID NO:21:DVQLQESGGGLVQAGGSLRLSCAASGRAFSVYAMAWFRQAPGKEREFVSSIAGSSSTYYTNYADSVKGRFTISRDNAKNTMYLQMDELKDEDTAVYYCAASPRSNNGRETRHYDYWGQGTQVTVSS It includes an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in or in SEQ ID NO:21.

[0139] The amino acid sequence of the hinge region of CD8 is SEQ ID NO:8: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD That is the case.

[0140] The amino acid sequence of the transmembrane region of CD8 is SEQ ID NO:9: IYIWAPLAGTCGVLLLSLVITLYC That is the case.

[0141] 2. Creation of mutant VSVG VSVG mutants 1, 2, 3, 4, 5, and 6, as well as a control group (VSVG mutant 1), were generated.

[0142] The ectodomain of the envelope glycoprotein (Indiana-VSVG) of the wild-type Indiana virus strain of the genus Becyclovirus contains the amino acid sequence shown in SEQ ID NO:1.

[0143] The full-length envelope glycoprotein of the aforementioned wild-type Indiana virus strain of the genus Becyclovirus contains the amino acid sequence shown in SEQ ID NO:25.

[0144] Regarding the control group VSVG1 (CTR-VSVG1), the ectodomain of CTR-VSVG1 contains the amino acid sequence shown in SEQ ID NO:26, in which the 354th amino acid of the amino acid sequence shown in SEQ ID NO:1 is substituted from arginine R to glutamine Q. In other words, compared to the wild-type Indiana-VSVG, the ectodomain of CTR-VSVG1 has the R354Q mutation.

[0145] Regarding VSVG mutant 1, the ectodomain of VSVG mutant 1 contains the amino acid sequence shown in SEQ ID NO:2, in which the 354th amino acid of the amino acid sequence shown in SEQ ID NO:1 is replaced from arginine R to glutamine Q, the 214th amino acid is replaced from threonine T to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A. In other words, compared to the wild-type Indiana-VSVG, the ectodomain of VSVG mutant 1 has the mutations T214N, T352A, and R354Q.

[0146] Regarding VSVG mutant 2, the ectodomain of VSVG mutant 2 contains the amino acid sequence shown in SEQ ID NO:3, in which the 354th amino acid of the amino acid sequence shown in SEQ ID NO:1 is replaced from arginine R to glutamine Q, the 214th amino acid is replaced from threonine T to asparagine N, the 352nd amino acid is replaced from threonine T to alanine A, the 50th amino acid is replaced from lysine K to threonine T, and the 146th amino acid is replaced from serine S to threonine T. In other words, compared to the wild-type Indiana-VSVG, the ectodomain of VSVG mutant 2 has the mutations K50T, S146T, T214N, T352A, and R354Q.

[0147] Regarding VSVG mutant 3, the ectodomain of VSVG mutant 3 contains the amino acid sequence shown in SEQ ID NO:11, in which the amino acid sequence shown in SEQ ID NO:11 has a deletion of lysine, which is the 47th amino acid of the amino acid sequence shown in SEQ ID NO:1, the 214th amino acid is replaced from threonine T to asparagine N (compared to before the deletion of K47), and the 352nd amino acid is replaced from threonine T to alanine A (compared to before the deletion of K47). In other words, compared to the wild-type Indiana-VSVG, the ectodomain of VSVG mutant 3 has the K47 deletion, T214N and T352A mutations.

[0148] Regarding VSVG mutant 4, the ectodomain of VSVG mutant 4 contains an amino acid sequence as shown in SEQ ID NO:12, and the amino acid sequence as shown in SEQ ID NO:12 is SEQ ID As shown in NO:1, the 47th amino acid, lysine, is deleted, the 214th amino acid (compared to before the K47 deletion) is replaced from threonine T to asparagine N, the 352nd amino acid (compared to before the K47 deletion) is replaced from threonine T to alanine A, the 50th amino acid (compared to before the K47 deletion) is replaced from lysine K to threonine T, and the 146th amino acid (compared to before the K47 deletion) is replaced from serine S to threonine T. In other words, compared to the wild-type Indiana-VSVG, the ectodomain of VSVG mutant 4 has the K47 deletion, K50T, S146T, T214N, and T352A mutations.

[0149] 3. Covering of delayed-onset viruses A. Prepare the following four plasmids and package the delayed viruses of VSVG mutant 1 (control group) and VSVG mutants 1, 2, 3, and 4. The above-mentioned delayed-onset virus possesses a mutant envelope plasmid containing a polynucleotide encoding the mutant VSVG and a polynucleotide encoding the membrane-bound CD7 single-domain antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the target plasmid pGClenti-GFP (GFP fluorescent protein), and the mutant VSVG envelope plasmid is synthesized by a conventional molecular cloning method. The mutant VSVG includes the control group VSVG or VSVG mutant 1, 2, 3, or 4. B. Package the slow-release viruses of VSVG from the control group (Delayed Virus 1 and VSVG mutants 1, 2, 3, and 4). The four plasmids are mixed together, and the four plasmids are used to infect the HEK-293T cell line, which is a packaging cell line, with a PEI reagent. The specific steps are: On day 0, 9 μg of the target plasmid, 4 μg of pMDLg / pRRE packaging plasmid, 2 μg of pRSV-REV packaging plasmid, and 2 μg of the aforementioned mutant envelope plasmid were added to 1 L of Opti-MEM medium (Opti-MEM alpha low serum medium, brand name: GIBCO, catalog number: #SP0272), shaken evenly, and then 64 μL of PEI reagent was added. After homogeneous mixing by pipetting, the mixture was allowed to stand for 10 minutes. Immediately thereafter, it was added to the HEK-293 T cell medium, the medium was refreshed after 6 hours, the supernatant was collected 48 hours after infection, filtered through a 0.45 μm filtration membrane, centrifuged at 50000 g for 2.5 hours, and the supernatant was aspirated and removed. The following are packaged: a slow-onset virus 1 of the control group VSVG whose viral envelope contains VSVG1 of the control group; a slow-onset virus of VSVG mutant 1 whose viral envelope contains VSVG mutant 1; a slow-onset virus of VSVG mutant 2 whose viral envelope contains VSVG mutant 2; a slow-onset virus of VSVG mutant 3 whose viral envelope contains VSVG mutant 3; and a slow-onset virus of VSVG mutant 4 whose viral envelope contains VSVG mutant 4. Using 200 μL of F12 medium, the VSVG delayed virus 1 and VSVG mutants 1, 2, 3, 4, 5, and 6 from the control group were resuspended and separated, and then stored frozen at -80°C.

[0150] HEK-293T cell medium is DMEM + 10% FBS. DMEM's brand name is GIBCO, and its part number is #C12430500BT. FBS is a brand with the name EXCELL and product number #FSP500. F12 culture medium has the brand name GIBCO and the catalog number #C11330500BT. The syringe filter is branded as SORFA and has part number #622120.

[0151] 4. Viral infection In some specific approaches, designing the first and second molecules involves mutating their amino acid sequences. Designing the first molecule, i.e., modifying the viral surface with an antibody / ligand that can target and identify a specific receptor, restricts its design and receptor binding. However, the mutant viral envelope glycoprotein, possessing endosomal / lysosome escape ability and complement inactivation ability, can rely on the first molecule as the second molecule. This allows the virus to target and infect target cells regardless of whether the target cell expresses the receptor for the viral envelope glycoprotein, thus significantly increasing the accuracy of targeting and not antagonizing the enhanced ability to be inactivated by serum complement.

[0152] On day 0, 1 × 10 of multiple groups 5 Each individual Jurkat cell (human T cell lymphocytic leukemia cell) was taken and resuspended in 200 μL of a Jurkat cell culture system containing complement (10% human plasma) and a Jurkat cell culture system without complement (10% human albumin HSA). Based on 10% human plasma or 10% human albumin, the Jurkat cell culture system contained 1640 medium (brand name: Elgbio, catalog number: TH80809) and 10% FBS.

[0153] The stock solutions of VSVG delayed virus 1 from the control group and the delayed viruses of VSVG mutants 1, 3, and 4 were each concentrated 200-fold. Then, 10 μL of the supernatant from each group's virus solution was taken and added to each of the Jurkat cell culture systems, either containing complement (10% human plasma) or not containing complement (10% human albumin HSA). The mixtures were then thoroughly mixed, left in 5% CO2, and cultured in a 37°C incubator. Flow cytometry was performed on the second day to measure GFP expression, and the results are shown in Figure 2. The Jurkat cell culture system contains 1640 medium (brand name: Elgbio, catalog number: TH80809) and 10% FBS. The measurement results for the CTR group represent the infection efficiency of VSVG delayed virus 1 from the control group infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. The measurement results for Group 1 represent the infection efficiency of the delayed-onset virus of VSVG mutant 1 infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. The measurement results for Group 2 represent the infection efficiency of the VSVG mutant 3's delayed-onset virus infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. The measurement results for Group 3 represent the infection efficiency of the delayed-onset virus of the VSVG mutant 4 infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. As can be seen from Figure 2, when complement is present, VSVG late-onset virus 1 from the control group, whose viral envelope glycoprotein does not possess the first mutation, is affected by complement inactivation, and its efficiency in infecting CD7+ Jurkat cells may decrease (compared to a medium without complement). However, if the late-onset viruses of VSVG mutants 1, 3, and 4, which were prepared using VSVG mutants 1, 3, and 4 whose viral envelope glycoproteins contain both the first and second mutations, are present, their efficiency in infecting CD7+ Jurkat cells is relatively unaffected by complement inactivation, even when complement is present. [Examples]

[0154] Packaging of multiple mutant VSVG slow-acting viruses Control groups VSVG2 and VSVG mutants 5, 6, and 7 were prepared.

[0155] Compared to the envelope glycoprotein (Cocal-VSVG) of the wild-type Cocal virus strain of the genus Becyclovirus, the ectodomains of VSVG mutants 5, 6, and 7 exhibit the first and second mutations. The ectodomain of the envelope glycoprotein of the aforementioned wild-type Cocal virus strain of the genus Becyclovirus contains the amino acid sequence shown in SEQ ID NO:10. The full-length envelope glycoprotein of the aforementioned wild-type Cocal virus strain of the genus Becyclovirus contains the amino acid sequence shown in SEQ ID NO:27.

[0156] Regarding the control group VSVG2 and the control group VSVG2 (CTR-VSVG2), the ectodomain of CTR-VSVG2 contains the amino acid sequence shown in SEQ ID NO:28, and in the amino acid sequence shown in SEQ ID NO:28, the 354th amino acid of the amino acid sequence shown in SEQ ID NO:10 is substituted from arginine R to glutamine Q. In other words, compared to the wild-type Cocal-VSVG, the ectodomain of CTR-VSVG2 has the R354Q mutation.

[0157] Regarding VSVG mutant 5, the ectodomain of VSVG mutant 5 contains the amino acid sequence shown in SEQ ID NO:18, in which the amino acid sequence shown in SEQ ID NO:18 has a deletion of lysine, which is the 47th amino acid of the amino acid sequence shown in SEQ ID NO:10, the 214th amino acid is replaced from lysine K to asparagine N (compared to before the deletion of K47), and the 352nd amino acid is replaced from threonine T to alanine A (compared to before the deletion of K47). In other words, compared to the wild-type Cocal-VSVG, the ectodomain of VSVG mutant 5 has K47 deletion, K214N and T352A mutations.

[0158] Regarding VSVG mutant 6, the ectodomain of VSVG mutant 6 contains the amino acid sequence shown in SEQ ID NO:20, in which the 354th amino acid of the amino acid sequence shown in SEQ ID NO:10 is replaced from arginine R to glutamine Q, the 214th amino acid is replaced from lysine K to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A. In other words, compared to the wild-type Cocal-VSVG, the ectodomain of VSVG mutant 6 has the mutations R354Q, K214N, and T352A.

[0159] Referring to the packaging method for packaging the delayed-onset viruses of VSVG mutants 1, 3, and 4 described in Example 1, the delayed-onset virus 2 of the control group VSVG, whose viral envelope contains the control group VSVG2, the delayed-onset virus of VSVG mutant 5, whose viral envelope contains the VSVG mutant 5, and the delayed-onset virus of VSVG mutant 6, whose viral envelope contains the VSVG mutant 6 were packaged, respectively.

[0160] CD7+ Jurkat cells were infected with the VSVG delayed virus 2 and VSVG mutants 5 and 6 from the control group, respectively. Referring to the infection method described in Example 1 for the delayed-release viruses of VSVG mutants 1, 3, and 4 to infect CD7+ Jurkat cells, the delayed-release viruses of VSVG mutants 5 and 6 from the control group were added to Jurkat cell culture systems containing complement (10% human plasma) or not containing complement (10% human albumin HSA) to infect CD7+ Jurkat cells. Flow cytometry was performed on day 2 to measure GFP expression, and the results are shown in Figure 3. The measurement results for the CTR group represent the infection efficiency of the VSVG delayed virus 2 from the control group infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. The measurement results for Group 1 represent the infection efficiency of the delayed-onset virus of the VSVG mutant 6 infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. The measurement results for Group 2 represent the infection efficiency of the delayed-onset virus of VSVG mutant 5 infecting CD7+ Jurkat cells in a medium containing human albumin (complement inactivated) and a medium containing human plasma (complement-containing), respectively. As can be seen from Figure 3, the infection efficiency of the delayed-onset virus of VSVG mutant 5, which possesses the first mutation K214N+T352A and the second mutation K47 deletion, was superior to that of the control group VSVG delayed-onset virus 2, which does not possess the aforementioned complement inactivation mutation, and the delayed-onset virus of VSVG mutant 6, which possesses the first mutation K214N+T352A and the second mutation R354Q.

[0161] The above description is merely a specific embodiment of the present invention; however, the scope of protection of the present invention is by no means limited thereto. Any modifications or substitutions that a person skilled in the art within the scope of the present invention can easily conceive should fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should conform to the scope of protection of the claims.

Claims

1. An envelope glycoprotein of a vesicular stomatitis virus having a first mutation and a second mutation, characterized in that the first mutation prevents the envelope glycoprotein from mediating complement-mediated inactivation, and the second mutation reduces the envelope glycoprotein's ability to be recognized by receptors.

2. The first mutation includes an amino acid sequence in the ectodomain of the envelope glycoprotein that is listed in SEQ ID NO:1, or an amino acid sequence that has at least 50% agreement with the amino acid sequence listed in SEQ ID NO:1, and the amino acid sequence is a) The amino acid located at position 214 of SEQ ID NO: 1, b) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 214 of SEQ ID NO:1, c) The amino acid located at position 352 of SEQ ID NO: 1, d) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 352 of SEQ ID NO:1, e) SEQ ID NO: The amino acid located at position 50 of 1, f) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to the 50th position of SEQ ID NO:1, g) The amino acid located at position 146 of SEQ ID NO: 1, h) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 146 of SEQ ID NO:1 The envelope glycoprotein according to claim 1, characterized in that it contains a mutation in at least one of the amino acids.

3. The envelope glycoprotein according to claim 2, characterized in that the mutation is an insertion, deletion, or substitution.

4. The aforementioned amino acid sequence is, a) The amino acid located at position 214 of SEQ ID NO: 1, b) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 214 of SEQ ID NO:1, c) The amino acid located at position 352 of SEQ ID NO: 1, d) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 352 of SEQ ID NO:1, e) SEQ ID NO: The amino acid located at position 50 of 1, f) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to the 50th position of SEQ ID NO:1, g) The amino acid located at position 146 of SEQ ID NO: 1, h) After performing optimal global alignment with SEQ ID NO:1, the amino acid at the position corresponding to position 146 of SEQ ID NO:1 The envelope glycoprotein according to claim 3, characterized by comprising the substitution of at least one amino acid among the following.

5. The envelope glycoprotein according to claim 4, characterized in that the amino acid sequence includes one or more combinations of mutations, namely a substitution at T214, a substitution at T352, a substitution at K50, and a substitution at S146.

6. The envelope glycoprotein according to claim 5, characterized in that the amino acid sequence includes one or more combinations of mutations in which the 214th amino acid is replaced from threonine T to asparagine N, and / or the 352nd amino acid is replaced from threonine T to alanine, and / or the 50th amino acid is replaced from lysine K to threonine T, and / or the 146th amino acid is replaced from serine S to threonine T.

7. The aforementioned amino acid sequence is, (1) Replacement of K214 and T352, (2) Replacement of K214, T352, K50 and S146 The envelope glycoprotein according to claim 4, characterized in that it contains one of the mutations.

8. The aforementioned amino acid sequence is, (1) The 214th amino acid is replaced from lysine K to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A. (2) The 214th amino acid is replaced from lysine K to asparagine N, the 352nd amino acid is replaced from threonine T to alanine A, the 50th amino acid is replaced from lysine K to threonine T, and the 146th amino acid is replaced from serine S to threonine T. The envelope glycoprotein according to claim 7, characterized in that it contains one of the mutations.

9. The aforementioned amino acid sequence is, (1) The 214th amino acid is replaced from threonine T to asparagine N, and the 352nd amino acid is replaced from threonine T to alanine A. (2) The 214th amino acid is replaced from threonine T to asparagine N, the 352nd amino acid is replaced from threonine T to alanine A, the 50th amino acid is replaced from lysine K to threonine T, and the 146th amino acid is replaced from serine S to threonine T. The envelope glycoprotein according to claim 6, characterized in that it contains one of the mutations.

10. The second mutation includes an amino acid sequence in the ectodomain of the envelope glycoprotein that is listed in SEQ ID NO:1, or an amino acid sequence that has at least 50% agreement with the amino acid sequence listed in SEQ ID NO:1, and the amino acid sequence is 1) SEQ ID NO: 1 Substitution / deletion located at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T3 Substitutions / deletions located at position 52, substitutions / deletions located at E353, substitutions / deletions located at R354, deletions of amino acids located at positions 1-18, deletions of amino acids located at positions 19-36, deletions of amino acids located at positions 37-51, deletions of amino acids located at positions 314-384, deletions of amino acids located at positions 321-374, deletions of amino acids located at positions 331-364, deletions of amino acids located at positions 344-354, deletions of amino acids located at positions 345-353, 2) After performing optimal global alignment with SEQ ID NO: 1, replace / delete at the position corresponding to H8 of SEQ ID NO: 1, replace / delete at the position corresponding to N9, replace / delete at the position corresponding to Q10, replace / delete at the position corresponding to K47, replace / delete at the position corresponding to K50, replace / delete at the position corresponding to A51, replace / delete at the position corresponding to S183, replace / delete at the position corresponding to S179, replace / delete at the position corresponding to N180, replace / delete at the position corresponding to I182, replace / delete at the position corresponding to M184, replace / delete at the position corresponding to Y209, replace / delete at the position corresponding to I347, and the position corresponding to T350 Substitutions / deletions located at the following positions: T352, E353, R354, deletions at positions corresponding to amino acids 1-18, deletions at positions corresponding to amino acids 19-36, deletions at positions corresponding to amino acids 37-51, deletions at positions corresponding to amino acids 314-384, deletions at amino acids 321-374, deletions at positions corresponding to amino acids 331-364, deletions at positions corresponding to amino acids 344-354, and deletions at positions corresponding to amino acids 345-353. An envelope glycoprotein according to any one of claims 1 to 9, characterized by containing a mutation in at least one amino acid among the following.

11. The aforementioned amino acid sequence is, 1) Substitutions located at H8, N9, Q10, K47, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, R354, deletions, deletions of amino acids at positions 1-18, deletions of amino acids at positions 19-36, deletions of amino acids at positions 37-51, deletions of amino acids at positions 314-384, deletions of amino acids at positions 321-374, deletions of amino acids at positions 331-364, deletions of amino acids at positions 344-354, deletions of amino acids at positions 345-353, 2) After performing optimal global alignment with SEQ ID NO: 1, substitutions are made at the position corresponding to H8 of SEQ ID NO: 1, substitutions are made at the position corresponding to N9, substitutions are made at the position corresponding to Q10, substitutions are made at the position corresponding to K47, deletions are made at the position corresponding to K47, substitutions are made at the position corresponding to K50, substitutions are made at the position corresponding to A51, substitutions are made at the position corresponding to S183, substitutions are made at the position corresponding to S179, substitutions are made at the position corresponding to N180, substitutions are made at the position corresponding to I182, substitutions are made at the position corresponding to M184, substitutions are made at the position corresponding to Y209, substitutions are made at the position corresponding to I347, substitutions are made at the position corresponding to T350, and substitutions are made at the position corresponding to T352. A substitution, a substitution at the position corresponding to E353, a substitution at the position corresponding to R354, a deletion at the position corresponding to R354, a deletion at the position corresponding to amino acids 1-18, a deletion at the position corresponding to amino acids 19-36, a deletion at the position corresponding to amino acids 37-51, a deletion at the position corresponding to amino acids 314-384, a deletion at the position corresponding to amino acids 321-374, a deletion at the position corresponding to amino acids 331-364, a deletion at the position corresponding to amino acids 344-354, a deletion at the position corresponding to amino acids 345-353 The envelope glycoprotein according to claim 10, characterized in that it contains a mutation in at least one of the amino acids.

12. The envelope glycoprotein according to claim 11, characterized in that the amino acid sequence includes one or more combinations of mutations, namely substitution / deletion of K47 and substitution / deletion of R354.

13. The envelope glycoprotein according to claim 12, characterized in that the amino acid sequence further comprises one or more combinations of mutations in which the 47th amino acid is replaced from lysine K to glutamine Q, the 354th amino acid is replaced from arginine R to glutamine Q, the 47th amino acid is deleted, and the 354th amino acid is deleted.

14. The aforementioned amino acid sequence is, (1) Substitution of K47, (2) Replacement of R354 The envelope glycoprotein according to claim 12, characterized in that it contains one of the mutations.

15. The aforementioned amino acid sequence is, (1) The 47th amino acid is replaced from lysine K to glutamine Q. (2) The 354th amino acid is replaced from arginine R to glutamine Q. The envelope glycoprotein according to claim 14, characterized in that it contains one of the mutations.

16. The second mutation is such that the ectodomain of the envelope glycoprotein contains an amino acid sequence, the amino acid sequence listed in SEQ ID NO: 10, and the amino acid sequence is Substitution or deletion at Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350 This includes deletions, substitutions or deletions located at T352, substitutions or deletions located at E353, substitutions or deletions located at R354, deletions of amino acids located at positions 1 to 18, deletions of amino acids located at positions 19 to 36, deletions of amino acids located at positions 37 to 51, deletions of amino acids located at positions 314 to 384, deletions of amino acids located at positions 321 to 374, deletions of amino acids located at positions 331 to 364, deletions of amino acids located at positions 344 to 354, and deletions of amino acids located at positions 345 to 353. After performing optimal global alignment with SEQ ID NO: 10, substitutions or deletions at the positions corresponding to Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, and S350 are replaced or deleted. Substitutions or deletions at the corresponding positions, substitutions or deletions at the position corresponding to T352, substitutions or deletions at the position corresponding to E353, substitutions or deletions at the position corresponding to R354, deletions at the positions corresponding to amino acids 1 to 18, deletions at the positions corresponding to amino acids 19 to 36, deletions at the positions corresponding to amino acids 37 to 51, deletions at the positions corresponding to amino acids 314 to 384, deletions of amino acids at positions 321 to 374, deletions at the positions corresponding to amino acids 331 to 364, deletions at the positions corresponding to amino acids 344 to 354, deletions at the positions corresponding to amino acids 345 to 353 An envelope glycoprotein according to any one of claims 1 to 9, characterized in that it contains a mutation in at least one of the amino acids.

17. The aforementioned amino acid sequence is, Substitutions located at Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350, T352, E353, R354, deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, 345-353. After performing optimal global alignment with SEQ ID NO: 10, substitutions are made at the position corresponding to Q8 of SEQ ID NO: 10, substitutions at the position corresponding to S9, substitutions at the position corresponding to Q10, substitutions or deletions at the position corresponding to K47, substitutions at the position corresponding to K50, substitutions at the position corresponding to A51, substitutions at the position corresponding to D183, substitutions at the position corresponding to A179, substitutions at the position corresponding to T180, substitutions at the position corresponding to V182, substitutions at the position corresponding to T184, substitutions at the position corresponding to Y209, substitutions at the position corresponding to I347, substitutions at the position corresponding to S350, and substitutions corresponding to T352. Substitutions at the position of E353, substitutions or deletions at the position of R354, deletions at the position of amino acids 1 to 18, deletions at the position of amino acids 19 to 36, deletions at the position of amino acids 37 to 51, deletions at the position of amino acids 314 to 384, deletions of amino acids at the position of amino acids 321 to 374, deletions at the position of amino acids 331 to 364, deletions at the position of amino acids 344 to 354, deletions at the position of amino acids 345 to 353 The envelope glycoprotein according to claim 16, characterized in that it contains a mutation in at least one of the amino acids.

18. The envelope glycoprotein according to claim 17, characterized in that the amino acid sequence includes one or more combinations of mutations, namely substitution / deletion of K47 and substitution / deletion of R354.

19. The envelope glycoprotein according to claim 18, characterized in that the amino acid sequence further comprises one or more combinations of mutations in which the 47th amino acid is replaced from lysine K to glutamine Q, the 354th amino acid is replaced from arginine R to glutamine Q, the 47th amino acid is deleted, and the 354th amino acid is deleted.

20. The envelope glycoprotein according to any one of claims 1 to 19, characterized in that the receptor is LDL-R.

21. The envelope glycoprotein according to any one of claims 1 to 20, characterized in that the ectodomain of the envelope glycoprotein contains one of the aforementioned amino acid sequences, such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:

20.

22. A nucleic acid molecule encoding an envelope glycoprotein according to any one of claims 1 to 21.

23. A vector comprising at least one nucleic acid molecule according to claim 22, or expressing an envelope glycoprotein according to any one of claims 1 to 21.

24. The envelope glycoprotein according to claim 23, characterized in that the vector is a slow-acting viral vector or a retroviral vector.

25. A targeting vector comprising a first molecule that binds to the endocytosis receptor of a target cell, and a second molecule that does not deactivate complement, but rather promotes the release of a substance carried by the targeting vector into the cytoplasm.

26. The targeting vector according to claim 25, characterized in that the second molecule promotes the generation of endosomal or lysosomal escape by the targeting vector and prevents the targeting vector from being inactivated by complement.

27. The targeting vector according to claim 26, characterized in that the second molecule is an envelope glycoprotein according to any one of claims 1 to 21.

28. The targeting vector according to any one of claims 25 to 27, characterized in that the targeting vector is an enveloped virus.

29. The targeting vector according to claim 28, characterized in that the enveloped virus is selected from a retroviral vector and a slow-release viral vector.

30. The targeting vector according to claim 29, characterized in that the delayed-release viral vector is a VSV vector.

31. The targeting vector according to claim 29 or 30, characterized in that the first molecule is not part of the viral envelope glycoprotein.

32. The targeting vector according to any one of claims 25 to 31, characterized in that the first molecule comprises a transmembrane peptide portion and an antibody or ligand that binds to the endocytosis receptor of a target cell.

33. The targeting vector according to claim 32, characterized in that the first molecule further comprises an extracellular hinge region.

34. The endocytosis receptors mentioned above are 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, and CD30. TROP2, FRα, STEAP1, ENPP3, GCC, SLC44A4, NaPi2b, CA9, SC-16, CD142, P-cadherin, PSMA, ED-B, endothelin receptor ETB, TN-C, collagen IV, periostin, CEACAM, c-MET, TDGF1, IGF1R, mesoserine, TIM1, NCAM1, ZIP6, CD166, GPNM A targeting vector according to claim 32 or 33, characterized by being selected from B, SDC1, sphingoglycolipid, TfR, ganglioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, and ASGPR.

35. The targeting vector according to claim 34, characterized in that the endocytosis receptor is CD7.

36. The antibody is an anti-CD7 single-domain antibody, and the HCDR1 domain of the anti-CD7 single-domain antibody contains an amino acid sequence as shown in SEQ ID NO: 22, or having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 domain of the anti-CD7 single-domain antibody contains an amino acid sequence as shown in SEQ ID NO: 23, or having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement with the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 domain of the anti-CD7 single-domain antibody is SEQ ID The targeting vector according to claim 35, characterized by comprising an amino acid sequence having a degree of agreement of at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence shown in NO:24 or SEQ ID NO:

24.

37. The targeting vector according to claim 36, characterized in that the anti-CD7 single-domain antibody (VHH) includes an amino acid sequence such as that shown in SEQ ID NO: 21, or such sequence has a degree of agreement of at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence shown in SEQ ID NO:

21.

38. The targeting vector according to any one of claims 25 to 37, characterized in that the substance is at least one of a small molecule compound, a protein, a polypeptide, RNA, and DNA.

39. The targeting vector according to claim 38, characterized in that the substance contains a polypeptide encoding a receptor that binds to an antigen.

40. The targeting vector according to any one of claims 25 to 39, characterized in that the target cells are lymphocytes, myeloid cells, hematopoietic stem cells / hematopoietic cells, or non-blood cells.

41. A method for preparing a targeting vector, comprising the steps of designing a first molecule based on the endocytosis receptor of a target cell, designing a second molecule, and preparing a targeting vector by loading the first molecule, the second molecule, and a substance to be carried by the vector, according to any one of claims 25 to 40.

42. The targeting vector according to claim 41, characterized in that designing the second molecule includes mutating the amino acid sequence of the second molecule.

43. Application of the targeting vector according to any one of claims 25 to 40 in the delivery of drugs or vaccines.

44. A targeting vector according to any one of claims 25 to 40, used for delivering small molecule compounds, proteins, polypeptides, RNA, or DNA.

45. A method for introducing a substance into cells, comprising bringing the cells into contact with the targeting vector described in any one of claims 25 to 40.

46. The method according to claim 45, wherein the cells are mammalian cells.

47. The method according to claim 45, wherein the cells are normal cells or cancer cells.

48. The method according to claim 45, wherein 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.

49. The method according to claim 45, wherein the contact is performed inside or outside the body.

50. A host cell comprising or expressing an envelope glycoprotein according to any one of claims 1 to 21, comprising a nucleic acid molecule according to claim 22, or comprising a targeting vector according to any one of claims 25 to 40.

51. A composition comprising an envelope glycoprotein according to any one of claims 1 to 21, a nucleic acid molecule according to claim 22, or a targeting vector, a host cell according to claim 50, or any combination thereof, according to any one of claims 25 to 40.

52. The composition according to claim 51, which is used as a pharmaceutical agent.

53. The composition according to claim 51, which is used in gene therapy, immunotherapy, cell therapy, treatment of genetic diseases, and treatment of cancer.

54. A method for treating a disease in which a subject is suffering or killing disease cells in a subject, characterized by administering to a subject the composition described in any one of claims 51 to 53.

55. The method according to claim 54, characterized in that the disease includes cancer, and the cancer includes blood cancer and solid tumors.

56. The method according to claim 54 or 55, characterized in that the administration is selected from at least one of the following: administration by mouth, nose, vein, peritoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intrafocal, sustained-release system, and implantable device.

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